Numerous therapeutic measures have been developed in oncology to combat various types of cancer, which is a leading cause of death worldwide. These therapies range from conventional approaches to high-end precision medicines, all aimed at ensuring therapeutic efficacy and the patient's survival. However, drug resistance and off-target side effects continue to hinder treatment efficacy. In the case of solid tumors that have hypoxic regions that cause treatment resistance and a high risk of tumor recurrence. To address this, advanced therapies have been designed or under pipeline to specifically target cancer cells by considering their unique features. This review article primarily focuses on different treatment methods developed using the peculiar properties of the tumor microenvironment.

Keywords: tumor microenvironment, targeted therapy, hypoxia, nanoparticles, radiation therapy, chemotherapy, combination therapy, radiosensitizer, photodynamic therapy, immunotherapy, hyperthermia

Tumors are unusual masses of tissues characterized by undisciplined growth and proliferation of cells: these can be benign (non-cancerous) and malignant (cancerous). Cancer is the second-leading cause of mortality in human beings and refers to a group of diseases characterized by the development of malignant abnormal cells that divide uncontrollably and have the ability to spread throughout the body. Apart from uncontrolled proliferation, the other characteristics of cancer are evasion of apoptosis, angiogenesis, invasion of tissues and metastasis to different locations in the body. The proliferation and metastasis are the main causes of cancer mortality. Detection of cancer at an early stage increases the chances of complete cure of the disease. In spite of the human efforts to eradicate this disease from ancient times and the present day’s spectacular achievements in science, technology and medicine, a definite cure for cancer is yet to be realized. There is a steady yearly increase in the number of new cases of cancer in both the developed and the developing countries.

Tumor - major treatment modalities

The available treatment modalities for cancer include – surgery, radiation therapy, chemotherapy, immune therapy, hormone therapy, gene therapy, stem cell therapy etc. Among these, surgery, radiation therapy, and chemotherapy are the most widely used.

Surgery: Surgery has been one of the major modalities of cancer treatment for many types of cancers especially when they are not spread to other parts of the body. It is also used to take biopsy samples for diagnosis and prediction of the stage of cancer. Combination of surgery with either chemotherapy or radiotherapy is often employed and found to be effective in curing cancer.

Radiation therapy: Radiation therapy or radiotherapy is the most important modality of cancer treatment. It is highly cost-effective and approximately 80% of all cancer patients require radiation therapy either for curative or palliative purposes.¹ The high-energy radiations deposit energy while passing through tissues causing ionizations to produce free radicals and damaging the vital biological targets - cellular DNA and membrane - resulting in the mortality of cancer cells.^2,3 Advances in imaging techniques, computerized treatment planning systems, radiation treatment machines (with improved X-ray production and treatment delivery), use of high-energy particles, etc. have contributed a great deal to the success of radiation therapy.¹ Due to rapid proliferation, the tumor cells overgrow their vascular supply, resulting in centrally necrotic and hypoxic regions where the cells are refractory to radiation. To overcome this problem, either cells of the tumor have to be sensitized to radiation by using hypoxic cell sensitizers or higher doses of radiation have to be used. Clinically use of higher doses of radiation is not possible as the normal cells, surrounding the tumor, are well perfused, vascularized and remain oxygenated, and therefore suffer more radiation damage. This necessitates the protection of the normal cells from radiation injury. Amifostin or ethyol is the only clinically approved compound available to protect normal cells.⁴ Number of compounds (radiosensitizers) have been synthesized and reported to enhance the efficacy of radiation therapy.⁵ Most of them completed preclinical studies successfully and failed in clinical trials due to toxicity to mammalian organisms. Also, several hypoxic cell sensitizers useful in radiotherapy of cancer are at different stages of clinical trials.⁶ The nitrotriazole compound, Sanazole or AK-2123 (Figure 1), is an effective hypoxic cell radiosensitizer. The hypoxic cell radiosensitizing property of this compound and its lack of toxicity made it attractive as an adjuvant in radiation therapy of cancer. It has successfully completed phase III clinical trials and is used in clinics along with radiation to treat different types of cancers such as head and neck, cervical cancer etc. as a hypoxic radiosensitizer.⁷

Figure 1 Chemical structure of Sanazole (Molecular Formula: C21H32N2O).

The treatment with Sanazole (SAN) enhanced radiation sensitivity in post-irradiated aerobic and anaerobic cells.⁸ In patients with advanced head and neck cancer, the administration of SAN increased the sensitivity to hyper fractionated radiation treatment.⁹ SAN has, also, the capability to enhance the therapeutic potential and reduce the effective dose of antineoplastic agents in comparison with the drug alone.^10–12 Konovalova et al.,¹³ revealed that the therapeutic concentration of Sanazole demonstrated anti-metastatic activity in tumor-bearing mice. AK2123 has been found to augment the antineoplastic activity of the chemotherapeutic agent Mitomycin C of multidrug resistant tumors.¹³ According to Schepetkin et al.,¹⁴ the bio-activation as well as the radiation and chemotherapy - sensitizing property of SAN could be due to the involvement of enzymes such as xanthine oxidase and microsomal NADPH/cytochrome p450 reductase.¹⁴ The ability of SAN to accumulate in tumors was first demonstrated by Murugesan et al.,¹⁵ via administering Technetium-99m labelled cyclam-sanazole to solid tumor-bearing animals. This study also revealed its potential in tumor imaging. Further, Das et al., explored the hypoxic tumor targeting capability of SAN.¹⁶ The mechanism of sensitization of hypoxic tumor by SAN¹⁷ is partially credited to its ability to induce increased DNA damage.¹⁶ In human lymphoma cells (U937), SAN found to cause Fas ligand-induced Caspase 8 dependent apoptosis with the down regulation of hsp70 protein.¹⁸

Chemotherapy: Chemotherapy is a major therapeutic strategy in medical oncology using chemical agents or drugs to destroy cancer cells. Based on the mechanism of action, these agents are categorized mainly into alkylating agents, anti-metabolites, anti-microtubule agents, inhibitors of topoisomerase, and cytotoxic antibiotics.

Alkylating agents: These agents can alkylate macromolecules such as proteins and nucleic acids, basically derived from Mustard gas used in World War I.¹⁹ They can damage genomic DNA, generate interstrand and intrastrand cross links in DNA resulting in inhibition of cell division (S-phase) and induce apoptosis. Other alkylating agents used in chemotherapy are cisplatin (Figure 2.1) and its derivatives (carboplatin and oxaliplatin), nitrosoureas, mitomycin, cyclophosphamide (Figure 2.2) etc.^19,20

Figure 2 Alkylating agents. 1. Cisplatin and 2. Cyclophosphamid.

Anti-metabolites: They are usually the analogues of building blocks of DNA and RNA; hence interfere with the synthesis of nucleic acids. The cell cycle dependent activity of these agents induces the programmed cell death, apoptosis. Methotrexate (Figure 3.1) is an inhibitor of the enzyme, dihydrofolate reductase which decreases the synthesis of pyrimidine bases via inhibiting the production of folate coenzymes. Fluorouracil (Figure 3.2) is a nucleoside analogue which induces programmed cell death.^20,21

Figure 3 Anti-metabolites. 1. Methotrexate and 2. 5-fluorouracil.

Cytotoxic antiobiotics: Anthracyclines and bleomycins are under this group. Anthracyclines (eg. Doxorubicin; figure 4.1) are isolated from bacterium Streptomyces peucetius.

Doxorubicin: (DOX) is a well-known potent chemotherapeutic agent (Figure 4.1) approved by Food and Drug Administration, widely used against a range of cancers such as leukaemia, sarcoma etc.²² DOX has the ability to fight with rapidly dividing cells, thereby decreases the tumor progression. The major mechanisms of action include DNA intercalation which prevents DNA replication and transcription, free radical generation, topoisomerase inhibition and apoptosis.²³ However, its therapeutic usage is limited only by its toxicity especially cardiotoxicity. DOX causes toxicity to cardiac tissues through the induction of oxidative stress,²⁴ reduced antioxidants,^25,26 altered heart-related expression of genes^27–29 and prevention of biomolecule synthesis.³⁰

The antibiotic actinomycin can also intercalate DNAs and prevents the expression of genes.³¹ Bleomycins (Figure 4.2) are obtained from Streptomyces verticillus, known to cause DNA intercalation; DNA strand breaks and free radical damage.³²

Figure 4 Cytotoxic antibiotics. 1. Doxorubicin and 2. Bleomycin.

Anti-microtubule agents and topoisomerase inhibitors: Most of these agents are plant-derived compounds. Several natural products have been tested as anticancer agents and some of them are in clinical trials. These products are usually the secondary metabolites of microorganisms and/or plants shows distinctive structural diversity which helps the organism to adapt to the various biological situations.^33,34 The major plants with clinically verified antineoplastic activity are Catharanthus roseus, Camptothecin acuminata, Cephalotaxus harringtonia, Podophyllum peltatum, Taxus brevifolia, Viscum album, Annona bullata, Onchrosia elliptica, Rhizoma zedoariae and Asmina triloba.³⁵

The plant-derived anticancer agents in clinical use are categorized mainly into a) the vinca alkaloids, b) the epipodophyllotoxin lignans, c) the taxane diterpenoids, and d) the camptothecin quinolone alkaloid derivatives. Apart from these, there are several plant derived molecules which have anticancer activities and are useful in chemoprevention as well as treatment of cancer.

Vinca alkaloids: Vincaleukoblastine (Vincristine; Figure 5.1), an alkaloid isolated from Vinca rosea Linn shows anti-tumor activity in mice-bearing transplantable tumor.³⁶ Johnson et al (1960) reported the potential of anticancer agent vincaleukoblastine in tumor control via obstructing the essential cellular metabolic pathways.³⁷ According to Svoboda et al.,³⁸ leurocristine (Vinblastine; Figure 5.2), another Vinca alkaloid, has a wide anticancer activity in human tumors.^38,39 These alkaloids are known as ‘spindle poisons’ as they can interact with the cellular receptor, tubulin and prevents its assembly.^40,41

Figure 5 Vinca alkaloids. 1. Vincristine and 2. Vinblastin.

Epipodophyllotoxin lignans: The podophyllotoxin (Figure 6.1), a non-alkaloid lignan found to have antineoplatic activity, is isolated from Phodophyllum species.⁴² It interacts with the enzyme topoisomerase II and prevents DNA unwinding as well as replication.⁴³

Taxane diterpenoids: Taxol (Paclitaxel; Figure 6.2), isolated from Taxus brevifolia, is the first compound having taxane ring showed anti-leukemic and anti-tumor activities.⁴⁴ The mechanism of action of Paclitaxel is mainly through the interaction with ‘tubulin’, which prevents the disassembly of mitotic spindle and hence cell division.⁴⁵

Figure 6 Structure of 1. Podophyllotoxin and 2. Paclitaxel.

Camptothecin quinolone alkaloid derivatives: Camptothecin (Figure 7), a quinolone alkaloid toxic to tumor cells, is extracted from Camptotheca acuminata. Topotecan and irinotecan, two analogues of Camptothecin clinically accepted as cancer chemotherapeutic agents, found to have anti-leukemic and inhibitory effect on tumors.⁴⁶ The mechanism of action is mainly through its interaction with the enzyme topoisomerase I.^47,48

Figure 7 Structure of Camptothecin.

Curcumin: It is the most studied phenolic compound with anticancer property derived from roots of Curcuma longa L. Phase I and phase II clinical trials revealed the therapeutic potential of curcumin against various tumors.^49,50

Flavopiridol: is a cytotoxic flavone in clinical trials found to induce cell death in human lung carcinoma cells⁵¹ and also found to induce p53-independent programmed cell death in small cell lung carcinoma cells.⁵² Lycopene is a tomato carotenoid shows a variety of biological functions, especially anticancer activities in various cancers, both in vitro and in vivo conditions.⁵³

Resveratrol: a phenolic compound, found to interfere with tumor initiation and the various steps of tumor progression.⁵⁴ The prevention of growth of hepato-cellular cancer cells by resveratrol revealed its anticancer potential.⁵⁵

Berberine: (BBN) (Figure 8) is an isoquinoline alkaloid derived from plants in Berberidaceae family such as Berberis vulgaris (barberry), B. aristata (tree turmeric), B. aquifolium (Oregon grape), Hydrastis canadensis, Coptis chinensis (golden thread) and Arcangelisia flava (Menispermaceae).⁵⁶ BBN is known to have anti-microbial, anti-helmintic, anti-viral and anti-inflammatory activities.^57–60 Recently, research has been focussing mainly on the antineoplastic activities of BBN. BBN might contribute anticancer activity through inhibiting the growth of Helicobacter pylori, an organism known to cause peptic ulcer and gastric cancer.^61,62 BBN also found to influence the activation of proto-oncogene to oncogene. The activation of proto-oncogene, c-Ki-ras2 contributes to oncogenic processes in human embryonic carcinoma cells - Tera 1 and Tera 2. BBN is found to influence the morphological differentiations of teratocarcinoma cells into neuronal cells through the negative regulation of c-Ki-ras2.⁶³ Since it was found that Activator Protein (AP-1) plays pivotal role in tumor progression.⁶⁴ BBN has an inhibitory effect on AP-1 activity as evidenced from a reporter gene assay done in human hepatoma cells.⁶⁵

Figure 8 Structure of BBN.

Mantena et al (2006a and b) reported that BBN inhibits the growth of human epidermoid carcinoma A431 cells through cell cycle arrest at G1 phase and apoptosis via regulating Cdki-Cdk-cyclin cascade, activation of caspase 3 and poly (ADP-ribose) polymerase (PARP).^66,67 BBN was also reported to induce apoptosis in U937 cells while it enhances cell death in melanoma B16 cell line through the induction of apoptosis.⁶⁸

Grisendi et al.,⁶⁹ reviewed the importance of Nucleophosmin/B23 in tumor progression as it was recognized as a potent tumor marker for several human tumors. The importance of the enzyme telomerase in cancer opens new avenues for developing strategies for cancer therapy.^70,71 BBN was shown to reduce the activity of telomerase and nucleophosmin/B23, and induce programmed cell death (apoptosis) in human leukemia HL-60 cells.⁷² In 1969, Krey and Hahn⁷³ reported that BBN can interact with DNA molecules. Later in 1981 Rungsitiyakorn et al.⁷⁴ showed the influence pH in the binding of BBN to DNA. According to Liu et al (2008), the p53-associated cell cycle arrest, apoptosis and DNA double strand breaks were induced in human osteosarcoma cells by the treatment with BBN.⁷⁵ This BBN is found to induce cell death through apoptosis in human colon cancer cells by various biochemical reactions.⁷⁶

Immunotherapy: Cancer cells express different molecules on its surface in order to enhance its proliferation. These molecules may be cancer antigens or carbohydrates. Immunotherapy is basically used to enhance the immune system by targeting these molecules on the surface, to kill cancer cells. The cell death mechanisms include antibody - dependent cell-mediated cytotoxicity (use antibodies to attack specific surface molecules), complement system (use blood proteins after antigen-antibody interaction), cell signalling (binding of antibodies initiates several signalling pathways to activate cell death mechanisms) and payload (antibody is conjugated with drug, toxin, small interfering RNA or radioisotope against antigen on the cell surface.^77–80 A combination of antibodies with radionuclides, radio-immunotherapy, has also been reported to be effective in cancer therapy.⁸¹

Photodynamic therapy: In this mode of treatment light-sensitive compounds, which are non-toxic to cells, have been used. When exposed to light these compounds become toxic. In the presence of light, photo sensitizers get excited and produce highly reactive free radicals, destroy the target cells. Some examples of photosensitizers are eg. aminolevulinic acid (natural), Allumera, Photofrin, Visudyne, Levulan etc. (commercially available). The application of photosensitizing agent to the body is purely depends on the part of the body being treated.⁸²

Hyperthermia: The controlled use of high body temperature than normal is often capable to wipe out cancer cells. Exposing the cells to higher temperature than normal body temperature, cause alterations and make the cells more likely to be affected by the treatments such as radiotherapy and chemotherapy. Hyperthermia can be used in two different ways.

• Local hyperthermia or thermal ablation: Very high temperatures are used to kill a small area of cells.
• Regional hyperthermia or whole-body hyperthermia: A part of the body or whole body is exposed to slightly higher body temperature to destroy the cells and helps other cancer treatments work better. In whole body hyperthermia, body heated to 39^oC to 43^oC to treat cancer cells.⁸³

Laser therapy: Laser, a narrow beam of light, has a single wavelength and can be used instead of scalpel in surgery. The commonly used types of lasers in these treatments are Carbon dioxide (CO₂), Argon, Nd:YAG (Neodymium: Yttrium-Aluminum-Garnet), Er:YAG (erbium: yttrium aluminum garnet); Ho:YAG (holium: yttrium aluminum garnet), copper vapor, and diode lasers [Lasers in Cancer Treatment. American Cancer Society].

• However, most of these treatment strategies are coupled with several harmful side effects and hence the therapeutic efficacy in terms of specificity gets compromised, causing damage to biological systems.⁸⁴ The treatments such as immunotherapy, laser therapy, hormone therapy and photodynamic therapy are still in the developmental stage. Most of the chemotherapeutic agents are known to cause systemic toxicities due to the lack of its therapeutic specificity. For example, the antineoplastic agents, Doxorubicin cause severe toxicity to heart tissues⁸⁵ and Cisplatin has been reported to generate renal injury in association with damages in tumor cells.⁸⁶

The unique feature of all tumors is the rapid proliferation of the cells. The therapeutic strategy for tumor is particularly directed towards the rapidly proliferating cells. Chemotherapeutics predominantly affect rapidly proliferating tumor cells through interfering cell division, metabolic processes in the cells or cause damage to vital cellular targets such as DNA and membrane. However, rapidly dividing normal cells, such as cells of the bone marrow, intestine and hair follicles are also affected. The therapeutic effectiveness of drug increases with increasing doses of administration. However, with increase in doses of administration there is an increase in systemic toxicities and side effects which compromises the therapeutic dose.

By targeted delivery of drugs specifically to the tumor, one can achieve maximum therapeutic efficacy without side effects. Drugs can be delivered directly in the tumor by intratumoral injection at the site.^87,88 This is possible only in case of peripheral tumors. For most other tumors other means of delivering drugs have to be adopted. Intratumoral injection of carrier-based chemotherapeutics has also been tried.⁸⁹ Delivery vehicles such as liposomes⁹⁰ and membrane sacs of red blood cell⁹⁰ have been tried and the success was limited. Thermo labile liposomes carrying the therapeutics are of great advantage,⁹¹ following administration, increasing the temperature at the tumor site can specifically release the contents in the tumor, while the liposomes remaining intact in other tissues. This would specifically destroy the tumor without affecting normal tissues.⁹² The recent upsurge in nanotechnology and nano-medicine has contributed to the development of elegant novel strategies for delivering drugs to the tumor.^93,94

Nanoparticles - the drug delivery vehicle

The nanoparticles or nanomaterials have distinctive optical, magnetic and electronic properties, and are capable to carry therapeutic or diagnostic agents. By utilizing these unique properties including their large surface-to-volume ratio, it is possible to develop new theranostic strategies. Since 1980s to the present, several technologies were implemented to enhance the activity and clinical success of nano-based therapeutics. The concept of PEGylation (poly ethylene glycol conjugation) was found to enhance the biopharmaceutical properties of proteins and biologically active substances.⁹⁵ The non-toxicity, water-solubility and less immunogenicity make PEG differ from other polymers.⁹⁶ ‘Active targeting’ of the drug can be achieved by conjugating the nanoparticles with specific ligand molecules for the cellular receptors, antibodies or peptides, providing specific interaction between receptors and ligands if they are in close proximity. Béduneau et al in 2007⁹⁷ demonstrated the effectiveness of active targeting by conjugating lipid nanocapsules of functionalized PEG with monoclonal antibodies against transferrin receptors (TFR) which are over expressed in cerebral epithelium, to facilitate specific drug delivery to the brain. Antibody-mediated cancer treatment was demonstrated by Daniels-Wells and Penichet (2016) using TFR-1 as a potent target.⁹⁸ Several PEGylated products are under various stages of clinical trials and some are in the clinic such as Doxil^® (liposome-doxorubicin product) and albumin-based nano-drug carriers (Abraxane^® - nanoparticle-albumin-paclitaxel and Albuferon-α^® - albumin and interferon-α).⁹⁹ The conjugation of a new targeting peptide SP90 with doxorubicin-encapsulated liposomes was found to enhance the therapeutic index by improving its accumulation in tumors and reducing the drug-induced systemic toxicities.¹⁰⁰ For tumor mitochondria specific photodynamic therapy, Wei et al.,¹⁰¹ developed a surface-modified Grapheme oxide based nano-drug in conjugation with the integrin αvβ3 monoclonal antibody.¹⁰¹

The passive targeting of nanoparticle-drug complexes is purely based on the phenomenon Enhanced permeability and Retention Effect. These nano-sized particles are entrapped by solid tumors because of their leaky blood vessels and inefficient lymphatic drainage system.^102,103 The size, shape, surface chemistry and stability¹⁰⁴ of nanoparticles have influence on its cellular uptake,¹⁰⁵ plasma clearance and bio-distribution.

The receptor-ligand interaction and subsequent downstream signalling cascade is influenced by the size of the particles. The gold and silver nanoparticles with size less than 100nm were effective to induce cell mortality.¹⁰⁶ The particles of size greater than 150nm will be cleared through reticuloendothelial system mediated by macrophage activation, while particle having size less than 10nm gets removed through renal clearance. Nanoparticles of the size 10-100nm will have good pharmacokinetic properties.

As the size of the particles increases above 150nm, the chance to get cleared from the circulation increases. The serum proteins get adsorbed on these particles coated with targeting molecules, prevents the accumulation of nano-complexes to solid tumors. The PEGylation of nanoparticles can improve the circulation time and passive targeting by inhibiting the interaction with serum proteins preventing macrophage - mediated plasma clearance.^107–109

The enhanced retention can further be improved by coating with cell specific targeting molecules. Several studies reported that the size and surface chemistry including surface charge of nanoparticles have a strong impact on targeted delivery and circulation time of the particles.^110,111 The smaller particles, in comparison with larger particles, can also penetrate deeply in to the tumor interstitium.¹¹²

Magnetic nanoparticles in targeted drug delivery

Nanoparticles can be of non-metallic or metallic origin. Non-metallic NPs constitutes natural carbohydrate polymers like chitin, chitosan, carrageenan, polylactic acid etc. Metallic NPs comprise oxides as well as salts of several metals including silver and gold. Nanoparticles containing paramagnetic elements such as iron, manganese etc. will have magnetic property and are called as magnetic nanoparticles. Iron-oxide nanoparticles - Fe₃O₄ and gamma Fe₂O₃ - are of special relevance. These are used in large number of studies for diagnostic and therapeutic purpose. Our tissues and blood do contain iron and iron-oxide nanoparticles are biocompatible. These NPs are cost-effective compared to several other nanoparticles of metals.

Magnetic iron-oxide nanoparticles (NP) have gained attention in cancer diagnosis (Imaging) and therapy (drug delivery). Because of the magnetic property, they can be directed to specific areas in the body by the application of an external magnetic field. Super-paramagnetic NPs can be used as contrast agents in magnetic resonance imaging of tumors (MRI).^113,114 Several magnetic NPs are in the clinic as contrast agents such as AMI-25 for liver/spleen imaging, AMI-227 for lymph node imaging (size is 20-40nm), NC100150 (Clariscan, size is 20nm) for perfusion imaging and NC100150 for MR angiography.¹¹⁵ NPs have been extensively employed as drug delivery vehicle in several studies. The importance of NPs in tumor therapy is mainly due to its capability to 1) transport and localize under the influence of an external magnetic field, 2) generate hyperthermia in the presence of alternating magnetic field and 3) carry targeting molecules to enhance active drug delivery.

Magnetic hyperthermia: IONP are capable of generating heat (hyperthermia) in the presence of an alternating magnetic field. The quantity of heat generated depends merely on the magnetic properties of the material and intensity of the magnetic field.^116,117 Under hyperthermia (40-46⁰C) cancer cells cannot survive, while normal cells are unaffected.¹¹⁸ The altered microenvironment makes the tumor cells more sensitive to higher temperatures with the exception of central nervous system (CNS).¹¹⁹ As CNS was found to be sensitive at temperature 40-43℃ for more than 6hrs, it is possible to treat tumor associated with CNS only under special conditions.¹²⁰ During hyperthermia (41.8℃), cytoplasmic heat shock protein 72 has been reported to over express on tumor cells, which is absent in normal cells.¹²¹

Magnetic drug targeting: The magnetic property has given more attention to magnetic NPs in solid tumor therapy as these particles can be attracted to a desired region with the application of magnetic field either external or internal. Hence, the non-specificity associated with conventional chemotherapy can be overcome by complexing them with magnetic NPs.¹²² For the drug targeting, these nanocarriers should be biocompatible, hydrophilic and non-toxic. As mentioned earlier in this review; the diameter, shape, surface charge, surface modifications and composition of the magnetic nanoparticles have influence on magnetic property and drug delivery. Ma et al.,¹²³ developed a targeting strategy to overcome the deleterious effects of conventional chemotherapeutic agent, doxorubicin (having systemic toxicities, particularly cardiotoxicity and hepatotoxicity) by conjugating it with spherical carbon magnetic nanoparticles of size 40-50nm.¹²³ Doxorubicin was specifically targeted to solid tumor by conjugating with NPs and application of magnetic field externally¹²⁴ in an animal model. The clinical application of magnetic targeting of 4'-epidoxorubicin, with an external magnetic field, was successfully demonstrated in patients with solid tumors,¹²⁵ proving the advantage of this technique over conventional therapy.

The tumors which are >2cm away from the periphery of the body, cannot be targeted by the application of an external magnetic field because the strength of magnetic field decreases with increase in distance.¹²⁶ Takeda et al.,¹²⁷ demonstrated the essential application of magnetic NP - drug targeting to tumors, which are deep in site, using super conducting magnets.¹²⁷ Use of magnetic field internally to target NP-drug complexes to tumors was also investigated. The NP was coated with doxorubicin and this complex was targeted to tumor by implanting magnet inside the desired region using laparoscopic technique.¹²⁸ However, this strategy of drug targeting was found to cause several problems while applying to human situations.¹²⁹

Magnetic nanoparticles in gene delivery: The magnetic NPs can also be used as a carrier for the delivery of nucleic acids to cells as nucleic acids can bind to magnetic IONPs.¹³⁰ This binding property has been exploited in the purification of DNA.¹³¹ This gene transfer-mediated by the magnetic nanoparticle is called magnetofection. Thus, both RNA and DNA can be conveniently transport using this technique. The magnetic NP–DNA complex can be taken up by cells and the genes can be expressed in the recipient cells. This technique is extremely useful technique for controlling gene expression by introducing antisense oligonucleotides¹³² and siRNA.¹³³ Nucleic acid can be conveniently attracted towards magnetic nanoparticle by coating them with polyethyleneimine having positive charge.¹³⁴ The nucleic acid complexed with NPs is taken up by the cells. Magnetofection can reduce time of transfection and the dose of the vector. The uptake of the complexes by the cells can be enhanced by the use of dynamic magnetic field through oscillating high intensity magnetic field. Recently, in vivo applications of the magnetofection with enhanced tissue penetration by oscillating magnetic field have been demonstrated.¹³⁵ The oscillating magnetic field imparts extra energy to the system which in turn results in particle uptake against external barriers. The underlying mechanism involves non-linear motion of the particles under the influence of the oscillating magnetic field facilitate tissue penetration, overcoming external barriers like muscle surrounding the tumor. Carbon nanotubes coated with nickel have also been found useful in transferring DNA to the cells under in vitro condition using magnetic field.¹³⁶ Oxidative therapy using complexes of magnetic NPs and D-aminoacid oxidase have been demonstrated in an animal model. This has proved even enzymes can be specifically targeted to tumor using magnetic NPs with the help of external magnetic field.^137,138

A new era in cancer therapy relies on precision medicine in which the treatment is tailored to the unique features of the tumor of an individual patient. This approach considers various factors such as genetics, lifestyle, and the unique features of cellular and molecular events of the tumor and its microenvironment. Here, tumor microenvironment plays a major role in customising therapy, and its inimitable features provide ample opportunities for designing diagnostic and therapeutic strategies. These features include an altered extracellular matrix, undeveloped leaky vasculature, hypoxia, acidity, lack of lymphatic drainage etc. which support cell proliferation and metastasis. This heterogeneity of tumor microenvironment together with genetic and epigenetic changes contributes to tumor progression and drug resistance.^139–141 Hence, a proper understanding of these interlinked characteristics of the tumor microenvironment, especially at its cellular and molecular levels is essential for better therapeutic gain. Figure 9 showcases the interconnected features of tumor microenvironment.

The (Figure 9.a) illustrates interlinked characteristic features of the tumor microenvironment. Figure 9.b depicts oxygen-dependent altered signalling pathway of Hypoxia-inducible factor-1α (HIF-1α): Under normoxic conditions, the enzyme with prolyl hydroxylase domain hydrolyses HIF-1α which promotes polyubiquitination by von Hippel-Lindau tumor suppressor protein (vHL). When the prolyl hydroxylase domain becomes inactive under a hypoxic environment and prevents hydroxylation, HIF-1α translocates to the nucleus, dimerizes with HIF-1β and recruits other factors. Ultimately, this promotes the binding of HIF-1α/ HIF-1β complexes to hypoxia-response elements (HRE) which leads to the activation of hypoxia-dependent downstream signalling pathways.

Figure 9 Showcases the interconnected features of tumor microenvironment.

Extracellular matrix (ECM): a dynamic contributor to tumor progression

The ECM is a complex network of highly cross-linked proteins that provide architectural support to the cells and control cellular activities in tissues. It is found in interstitial forms within organs and in specialized forms such as basement membranes and vascular endothelium, surrounding certain tissues and cell types. The ECM comprises proteoglycans and several fibrous proteins, mainly collagens- most abundant, elastin, fibronectins and laminins.¹⁴² Changes in the organization of ECM are the hallmark of cancer progression, initiating a rewiring of cellular and molecular signalling that nourishes tumor progression. It includes altered integrin signalling and collagen degradation. Along with collagen deposition, the overproduction of lysyl oxidase contributes to ECM stiffness and the loss of its integrity, altering the downstream signalling cascade and promoting tumor progression.^143,144

Heterogeneity in tumor vasculature is the basis for unique physiology

In normal tissue systems, the vasculature is hierarchically organized and regulated by the balanced expression of pro-angiogenic and anti-angiogenic factors based on metabolic demand. However, in tumors, the aggressive growth and proliferation of cells demand the hyper-activation of pro-angiogenic factors, leading to the improper development of vascular networks. The blood vessels in tumor tissues are heterogeneous and contain both normal blood vessels from which tumor cells invade and tumour-induced microvasculature. These blood vessels are often dilated and convoluted and have a branching pattern entirely different from normal vasculature. The altered endothelial linings, defective basement membranes and loss of pericytes make them leaky. Functionally, the immature blood vessels interrupt the proper supply of oxygen and nutrients to tumor cells, and the absence of functional lymphatic drainage hinders the clearance of waste materials from tumor mass.^145,146

Tumor hypoxia and acidity

Tumor hypoxia is a medical condition where tissue oxygen levels are significantly lower than normal. This is caused by the rapid cell proliferation and growth of tumors, which restricts the invasion of blood vessels into the tumor. Cells far away from functional blood vessels are presumed necrotic, while cells in the hypoxic region are viable but have a lower proliferation rate and/or are in the resting G0 phase of the cell cycle.^147,148 Further nutrient deprivation in hypoxic cells can lead to the migration of hypoxic tumor cells into the necrotic zone. As the tumor progresses, this process continues and helps to maintain tumor survival. Most anticancer therapies are primarily effective against rapidly proliferating cells, which means that hypoxic cells often escape treatment.¹⁴⁹ The cells in the normoxic region that are dividing rapidly are highly exposed to treatments and undergo cell death, leading to improved nutrient supply to the previously hypoxic cells, allowing them to divide rapidly to regenerate the tumor. Oxygen availability plays a crucial role in the effectiveness of radiation therapy. Research has shown that radiation damages cancerous cells depending on oxygen availability during irradiation.^150–152 Therefore, hypoxic regions in solid tumors require higher doses of radiation for mortality, making them more resistant to treatment. Tumor cells follow an anaerobic glycolytic pathway for energy generation which contributes to the greater demand for nutrients for the uncontrolled rapid growth and proliferation of tumors.^153,154 Due to the absence of a proper lymphatic drainage system, the major by-products of this metabolic pathway accumulate in hypoxic tumor cells resulting in tumor acidity.¹⁵⁵

Hypoxic environment in tumor: a feasible target for tumor therapy

Solid tumors have altered diffusion geometry that results in reduced oxygen and nutrient supply, leading to the formation of a hypoxic environment. The cells in the hypoxic zone, which are alive but in a quiescent phase (G0), promote malignancy and therapeutic resistance by activating genes responsible for tumor growth and metastasis.¹⁵⁶ Among them, hypoxia-inducible factor-1 (HIF-1) plays a crucial role in hypoxia-induced tumor progression and metastasis.¹⁵⁷ HIF-1 was identified by the recognition of the hypoxia response element (HRE) in the 3’ enhancer region of the gene erythropoietin, a hormone involved in erythrocyte proliferation and undergoes hypoxia-induced transcription.^158,159 The heterodimer HIF-1 comprises a cytoplasmic subunit HIF-1α and a nuclear subunit HIF-1β. The oxygen-dependent regulation of HIF-1α is schematically presented in Figure 9.b.

The best mechanism behind the regulation of HIF-1α protein is mediated by the von Hippel-Lindau (vHL) protein. In the presence of oxygen, vHL protein recruits HIF-1α for ubiquitination via the 26S proteasome degradation pathway. Prolyl-4-hydroxylase (PHD) hydoxylates prolyl residues in HIF-1α, promoting the binding of HIF-1α with vHL. However, in hypoxic conditions, tricarboxylic acid cycle intermediates such as succinate and fumarate, or mitochondrial reactive oxygen species inhibit PHD activity and stabilize HIF-1α. The accumulated HIF-1α in association with the nuclear subunit HIF-1β binds to the HRE in target genes that promote hypoxia-responsible gene expression.^160,161

The promoter region of vascular endothelial growth factor (VEGF), an important angiogenic factor - contains the HRE which is a binding site for HIF-1. This binding initiates VEGF expression which in turn promotes angiogenesis and subsequent tumor progression.¹⁶² In addition, the hypoxia in tumor milieu enhances the expression of epidermal growth factor receptors (EGFR). The EGFR signalling is known to enhance cell proliferation, prevent apoptosis and promote tumor angiogenesis and metastasis.^163,164

Selective therapy using hypoxia-activated pro-drug

There are two main types of drugs that target tumor hypoxia: quinone-based bio-reductive alkylating agents, such as mitomycin-C,¹⁶⁵ and nitroimidazole hypoxic cell radiosensitizers, such as Misonidazole.^166–168 In 1972, Lin et al. discovered that derivatives of benzoquinone can slow down the growth of adenocarcinoma 755 ascites cells and extend the lifespan of tumor-bearing mice. These compounds undergo alkylation after bio-reduction in hypoxic conditions.¹⁶⁹ In 1986, Zeman et al. proposed that the compound 3-amino-1,2,4-benzotriazine-1,4 dioxide (tirapazamine) can effectively kill cancer cells under hypoxic conditions as a selective bio-reductive agent.¹⁷⁰ Wilson pointed out the drawbacks of currently available hypoxia-selective cytotoxins as they are designed to destroy hypoxic cells which comprise a small fraction of the tumor population.¹⁷¹ Therefore, it necessitates combination treatments with either radiation, chemotherapy or photodynamic therapy to eliminate the tumor population.

To address challenges, researchers created diffusible cytotoxins, such as nitro-deactivated aromatic mustards and cobalt (III) complex-deactivated aliphatic mustards, which have bio-reduction potential. These nitrogen mustards can cause DNA damage but with less cell cycle specificity. They become activated when reacting with reductive triggers like nitro and Co (III) in hypoxic cells. By exploring the use of hypoxia in its bio-reduction, most of the metabolic transformations of drugs were prevented in normoxic cells.¹⁷² These drugs can become active through reductive metabolism in low-oxygen environments, producing toxic byproducts. The key mechanism behind this is that oxygen-sensitive enzymes, such as cytochrome p450 reductase, generate free radicals when reacting with these drugs. These free radicals can be toxic to cells, resulting in cell death.^173,174 However, when these drugs are metabolized by NAD(P)H dependent quinone oxidoreductase, which is not oxygen-sensitive, the two-electron product generated is less toxic in hypoxic cells than the one-electron product. In normoxic conditions, the free radicals generated through one-electron reduction react with oxygen to form superoxide radicals,¹⁷⁵ which are less toxic than the free radicals generated in hypoxic conditions.

Tirapazamine (TPZ) has been discovered to be cytotoxic in hypoxic conditions by Brown and Lee and Wilson.^173,176 The significance of TPZ (SR-4233) has been mainly attributed to its ability to enhance cytotoxicity in hypoxic mammalian cells¹⁷⁰ and improve toxicity to radiation. TPZ treatment, when combined with a well-known chemotherapeutic agent, Cisplatin, can enhance its anti-tumor potential in a hypoxia-dependent manner.^176,177 The therapeutic potential of TPZ has mainly been attributed to its protonated form in low-oxygen conditions.¹⁷³ However, recent evidence suggests that this protonated form is not the final toxic product. Rather, new radicals either hydroxyl or benzotriazinyl are involved.^178–180 Another pro-drug is Anthraquinone (AQ4N) which is activated to a hypoxia-selective cytotoxin through an unusual two-electron reduction mechanism achieved by the CYP3A members of the cytochrome P450 family.^181,182

Hypoxia-selective gene therapy

Tumor-specific proteins that arise due to differential gene expression provide characteristic properties to tumor cells. This presents an opportunity for controlling tumors through gene-targeting methods. A new gene-directed enzyme prodrug therapy approach has been developed to deliver the genes of specific enzymes with promoters containing hypoxia-response elements. Once the gene transfer takes place, the genes are transcribed and translated to produce the required enzymes that convert a pro-drug to an active cytotoxic drug.¹⁸³ There are some clinical trials of this approach where the genes of enzymes that can activate prodrugs into cytotoxins are introduced into tumor cells. In 2003, Binley et al. developed an optimized hypoxia-responsive promoter in adenoviral vectors that stimulates the hypoxia-targeted expression of human cytochrome (CYP2B6).¹⁸⁴ This promoter interrupts tumor growth by activating prodrugs to active cytotoxic agents. Some of the most interesting enzyme/prodrug gene therapies are combinations of herpes simplex-1 virus thymidine kinase/ganciclovir and cytosine deaminase/5-fluorocytosine.¹⁸⁵

Another approach in gene-directed enzyme pro-drug therapy involves transfecting a gene that encodes a one-electron reductase like cytochrome P450 3A4 for the hypoxia-specific activation of pro-drug AQ4N.¹⁸⁶ However, a significant challenge in this therapeutic approach is targeting the delivery of the enzyme/pro-drug to cells with high HIF-1 or hypoxia. An alternative strategy is to use a hypoxia-assisted adenoviral vector to transfect a reporter gene or a gene encoding the enzyme to human macrophages that can effectively activate the pro-drug as a cytotoxin.^187,188

Interfering HIF-1 activity

Hypoxia-induced tumor growth can be prevented by interfering with the HIF-1 activity. Inhibiting HIF-1 can stop the major adaptive responses of tumor progression. Several approaches have been developed to regulate the potential of HIF-1. One method is inhibiting the HIF-1-associated transactivation of genes, like VEGF and epidermal growth factors, to prevent hypoxia-induced tumor growth. Kung et al. (2000) discovered that interrupting the interaction between HIF-1 and its co-activators p300/CREB with a polypeptide hindered the transcription of target genes involved in tumor progression.¹⁸⁹ Another approach is inhibiting the HIF-1 protein via translation or destabilization inhibition. Geldanamycin enhances the degradation of HIF-1 protein, which reduces its level in the tumor and leads to tumor regression.¹⁹⁰ The intratumoral introduction of an antisense HIF-1α-containing plasmid led to the downregulation of VEGF, which reduced microvessel density. Although this treatment did not show any effect on T cell-mediated immunity, it synergized B cell-, NK cell-, and CD8 T cell-dependent cure of tumors. The antisense HIF-1α gene therapy thus enhanced the efficacy of immunotherapy¹⁹¹ and improved the therapeutic efficacy of doxorubicin.¹⁹² There have been significant advancements in the development and application of HIF-1α inhibitors for controlling tumors. Studies have shown that the chemotherapeutic drug cisplatin can enhance HIF-1α degradation in ovarian cancers that are sensitive to cisplatin.¹⁹³ An antisense oligonucleotide, identified as EZN-2698, has successfully completed a phase I clinical trial in patients with advanced solid tumors.^194,195 Also, a HIF-1 inhibitor called topotecan has completed clinical trials in non-small cell lung cancer.¹⁹⁵ Furthermore, the anti-tumor activity of gemcitabine has been demonstrated to induce immunogenic cancer cell death in pancreatic ductal adenocarcinoma by inhibiting HIF-1α through PX-478.¹⁹⁶

Therapy with anaerobic bacteria

Anaerobic bacterial therapy is relevant due to the necrotic regions in tumors where blood supply and oxygen are absent. The use of bacterial therapy has been documented for over a hundred years,¹⁹⁷ and active research is still being done in this area. Recent developments in this strategy involve targeting therapeutic agents to anaerobic necrotic areas in the tumor using a genetically engineered non-pathogenic strain of the bacterial genus Clostridium that can grow and localize in these regions.^198,199 The major approaches in bacterial therapy include using bacteria to enhance the therapeutic potential of drugs, as carriers of anti-neoplastic drugs, and as vectors in gene therapy.²⁰⁰ However, the most favorable approaches are the use of genetically modified bacterial strains for tumor destruction and bacterial gene-directed enzyme/pro-drug therapy. The gene encoding cytosine deaminase, an enzyme present in Escherichia coli that can convert pro-drug 5-fluorocytosine to the cytotoxic chemotherapeutic agent 5-fluorouracil, was cloned in Clostridium using an expression vector. This method could increase the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine several-fold.²⁰¹ Gardlik et al. reviewed the approach of Bacteria-mediated anti-angiogenesis therapy in tumor tissues.²⁰²

Use of more than one therapeutic agents or drugs has shown to be great advantage in many instances as combination of drugs with different mechanisms of action provide synergism in cancer therapy which could also prevent the treatment associated multi-drug resistance. Highly potent combinations of drugs are quite often associated with deleterious effects due to toxicities and side effects. Nanoparticle combinations of drugs are an alternative to overcome these deleterious effects.²⁰³ Single nanoparticle combinations of multiple drugs have great advantage over combined administration nanoparticle – single drug combinations since the former offers uniformity of the vehicle size, proper loading of drugs in desired proportion and time-dependent time release.

Nanoparticle platforms such as liposomes, dendrimers, polymeric nanoparticles etc. are employed in many instances for the co-delivery of chemotherapeutic drugs, siRNA, sensitizers etc. for tumor control.^204,205 Specific targeting of neoplastic drug doxorubicin to tumors have been achieved by complexing them with nanoparticles of iron-oxide and silver-oxide together with a hypoxic sensitizer Sanazole.^206,207 Targeting of the tumor by external magnetic field is effective in case of peripheral accessible tumors but this method is not suitable for deep seated internal tumors and for these chemotargeting could be effective. Chemotargeting of cytotoxic drugs to tumor could be achieved by utilizing the peculiar properties of tumour microenvironment, particularly the hypoxia in malignant tumors. Following administration, aromatic nitro compounds such as nitroimidazoles and nitrotrazoles get accummulated tumour bearing animals get accumulated in the hypoxic tumor regions in tumor bearing animals. This property of the nitrocompounds to get accumulated in tumor microenvironment is best utilized for chemo-targeting of therapeutics to the tumor tissiue. In tumour bearing mice, oral administration of a complex containing chemotherapeutic doxorubicin (DOX) or the cytotoxic phytoceutical, berberine (BBN) along with iron oxide nanoparticles (NP) and the nitrotriazole compound, sanazole (SAN) result in accumulation of the nanocomplexes (NP-DOX-SAN) and (NP-BBN-SAN) specifically in the tumor site resulting in reduction of tumor volumes in tumor bearing mice.^206–208 Thus nanoparticles can be effectively used for chemo-targeting cytotoxic drugs to cancer cells and achieve better therapeutic outcome. However, more studies are needed to initiate clinical trials.

The tumor specific proteins arising from differential expression of genes confer characteristic properties to tumor cells and provide opportunity for developing strategies of tumor control through gene targeting methods. Tumor cells in general display reduced apoptosis and if one can enhance the apoptosis, either by reducing the expression of anti-apoptotic proteins or increasing the expression of pro-apoptotic proteins, will have therapeutic benefit. Tumor hypoxia and associated expression of genes enhances tumor progression. Developing siRNA or antisense RNA techniques for the inhibition of expression of these genes (vegf, egfr, etc.) can be thought of as therapeutic strategy for tumor control. Certain killer peptides/proteins are expressed in cells which are in severe stress and trauma.²⁰⁹ These peptides trigger pathways to induce cell death. Tumor can be controlled by specific targeting of killer peptides to them using any of the nanocarriers. Thus, nanotechnology and the characteristics of tumor microenvironment can be harnessed to develop suitable strategies for tumor control.

None.

The authors declare that there is no conflict of interest.

None.

1. Bernier J, Hall EJ, Giaccia A. Radiation oncology: a century of achievements. Nat Rev Cancer. 2004;4(9):737–747.
2. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461(7267):1071–1078.
3. Lomax ME, Folkes LK, O'Neill P. Biological consequences of radiation-induced DNA damage: relevance to radiotherapy. Clin Oncology. 2013;25(10):578–585.
4. Nair CKK, Parida DK, Nomura T. Radioprotectors in radiotherapy. J Radiat Res. 2001;42(1): 21–37.
5. Sheehan JP, Shaffrey ME, Gupta B, et al. Improving the radiosensitivity of radioresistant and hypoxic glioblastoma. Future Oncol. 2010;6(10):1591–601.
6. Coleman CN, Bump EA, Kramer RA. Chemical modifiers of cancer treatment. J Clin Oncol. 1988;6(4):709–733.
7. Huilgol NG, Nair CKK, Kagia VT, editors, Radio sensitizers- a contemporary audit, New Delhi: Narosa publishing house; 2000.
8. Imamura M, Edgren M, Murata T, et al. Radiosensitization with a 3-nitrotriazole (ak-2123). Int J Oncol. 1995;6(4):841–845.
9. Huilgol NG, Chatterjee N, Mehta AR. An overview of the initial experience with AK-2123 as a hypoxic cell sensitizer with radiation in the treatment of advanced head and neck cancers. Int J Radiat Oncol Biol Phys. 1996;34(5):1121–1124.
10. Konovalova NP, Diatchkovskaya RF, Volkova LM, et al. Radiosensitizer AK-2123 as modulating agent in the chemotherapy of experimental metastases. Neoplasma. 1995;42(3):119–122.
11. Rao BS, Devi PU. Multimodality treatment using AK-2123, hydralazine, radiation & hyperthermia on a transplantable mouse tumour. Indian J Med Res. 1996;104:182–189.
12. Goncharova SA, Raevskaia TA, Konovalova NP, et al. The radiosensitizer AK-2123 enhances sensitivity of MDR-tumors to Mitomycin C. Vopr Onkol. 2000;46(2):202–208.
13. Konovalova NP, Volkova LM, Tatyanenko LV, et al. Inhibitory effect of radiosensitizer AK-2123 on experimental hepatic metastases and Ca2+ active transport. Neoplasma. 1997;44(6):361–365.
14. Schepetkin IA, Cherdyntseva NV, Kagiya VT. Sanazole as substrate of xanthine oxidase and microsomal NADPH/cytochrome P450 reductase. Pathophysiology. 2001;8(2):119–127.
15. Murugesan S, Shetty SJ, Noronha OP, et al. Technetium-99m-cyclam AK 2123: a novel marker for tumor hypoxia. Appl Radiat Isot. 2001;54(1):81–88.
16. Das T,  Chakraborty S, Banerjee  A, et al , Preparation and preliminary evaluation of a 177Lu – labeled sanazole derivative for possible use in targeting tumor hypoxia.  Biorg.Med.Chem. 2004:12(23):6077–6084.
17. Pasupathy K, Nair CK, Kagiya TV. Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae. J Radiat Res. 2001;42(2):217–227.
18. Yu DY, Zhao QL, Wei ZL, et al. Enhancement of hyperthermia-induced apoptosis by sanazole in human lymphoma U937 cells. Int J Hyperthermia. 2009;25(5):364–373.
19. Siddik ZH. Mechanisms of Action of cancer chemotherapeutic agents: DNA-interactive alkylating agents and anti-tumour platinum-based drugs. John Wiley & Sons Ltd; 2009.
20. Lind MJ. Principles of cytotoxic chemotherapy. Medicine. 2008;36(1):19–23.
21. Parker WB. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev. 2009;109(7):2880–2893.
22. Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the ugly effect. Curr Med Chem. 2009;16(25):3267–3285.
23. Minotti G, Menna P, Salvatorelli E, et al. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev. 2004;56(2):185–229.
24. Doroshow JH. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res. 1983;43(2):460–472.
25. Doroshow JH, Locker GY, Baldinger J, et al. The effect of doxorubicin on hepatic and cardiac glutathione. Res Commun Chem Pathol Pharmacol. 1979;26(2):285–295.
26. Olson RD, MacDonald JS, van Boxtel CJ, et al. Regulatory role of glutathione and soluble sulfhydryl groups in the toxicity of Adriamycin. J Exp Ther. 1980;215(2):450–454.
27. Kim Y, Ma A, Kitta K, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol. 2003;63(2):368–377.
28. Aries P, Paradis P, Lefevre C, et al. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci USA. 2004;101(18):6975– 6980.
29. Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis. 2004;49(5):330–352.
30. Odom AL, Hatwig CA, Stanley JS, et al. Biochemical determinants of Adriamycin toxicity in mouse liver, heart and intestine. Biochem Pharmacol. 1992;43(4):831–836.
31. Sobell HM. Actinomycin and DNA transcription. Proc Natl Acad Sci USA. 1985;82(16):5328–31.
32. Dorr RT. Bleomycin pharmacology: mechanism of action and resistance, and clinical pharmacokinetics. Semin Oncol. 1992;19(5):3–8.
33. Bindseil KU, Jakupovic J, Wolf D, et al. Pure compound libraries; a new perspective for natural product based drug discovery. Drug Discov Today. 2001;6(16):840–847.
34. Firn RD, Jones CG. Natural products - a simple model to explain chemical diversity. Nat Prod Rep. 2003;20(4):382–391.
35. Ram VJ, Kumari S. Natural products of plant origin as anticancer agents. Drug News Perspect. 2001;14(8):465–482.
36. Cutts JH, Beer CT, Noble AL. Biological properties of Vincaleukoblastine, an alkaloid in Vinca rosea Linn, with reference to its antitumor action. Cancer Res. 1960;20:1023–1031.
37. Johnson IS, Wright HF, Svoboda GH, et al. Antitumor principles derived from Vinca rosea Linn. Cancer Res. 1960;20:1016–1022.
38. Svoboda GH. Alkaloids of Vinca rosea (Catharanthus roseus). IX. Extraction and characterization of leurosidine and leurocristine. Lloydia. 1961;24:173–178.
39. Neuss N, Corman M, Boaz HE, et al. Vinca alkaloids. XI. Structures of leurocristine and vincaleukoblastine. J Am Chem Soc. 1962;84(8):1509–1510.
40. Guéritte-Voegelein F, Guénard D, Potier P. Anticancer substances of vegetable origin. Spindle poisons: vincaleukoblastine, leurocristine and navelbine; taxol and taxotere. C R Seances Soc Biol Fil. 1992;186(5):433–440.
41. Jordan A, Hadfield JA, Lawrence NJ, et al. Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle. Med Res Rev. 1998;18(4):259–296.
42. Xu H, Lv M, Tian X. A review on hemisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: 2003-2007. Current Med Chem. 2009;16(3):327–349.
43. Canel C, Moraes RM, Dayan FE, et al. Molecules of interest: Podophyllotoxin. Phytochem, 2000;54(2):115–120.
44. Wani MC, Taylor HL, Wall ME, et al. Plant antitumor agents. VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc. 1971;93(9):2325–2327.
45. Brito DA, Yang Z, Rieder CL. Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied. J Cell Biol. 2008;182(4):623–629.
46. Wall ME, Wani MC, Cook CE, et al. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc. 1966;88(16):3888–3890.
47. Liu LF, Desai SD, Li TK, et al. Mechanism of action of camptothecin. Ann N Y Acad Sci. 2000;922:1–10.
48. Ulukan H, Swaan PW. Camptothecins: a review of their chemotherapeutic potential. Drugs. 2002;62(14):2039–2057.
49. Bar-Sela G, Epelbaum R, Schaffer M. Curcumin as an anti-cancer agent: review of the gap between basic and clinical applications. Curr Med Chem. 2010;17(3):190–197.
50. Ji JL, Huang XF, Zhu HL. Curcumin and its formulations: potential anti-cancer agents. Anticancer Agents Med Chem. 2012;12(3):210–218.
51. Bible KC, Kaufmann SH. Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res. 1996;56(21):4856–4861.
52. Shapiro GI, Koestner DA, Matranga CB, et al. Flavopiridol induces cell cycle arrest and p53-independent apoptosis in non-small cell lung cancer cell lines. Clin Cancer Res. 1999;5(10):2925–2938.
53. Bhuvaneswari V, Nagini S. Lycopene: a review of its potential as an anticancer agent. Curr Med Chem Anticancer Agents. 2005;5(6):627–635.
54. Aggarwal BB, Bhardwaj A, Aggarwal RS, et al. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 2004;24(5A):2783–2784.
55. Bishayee A, Politis T, Darvesh AS. Resveratrol in the chemoprevention and treatment of hepatocellular carcinoma. Cancer Treat Rev. 2010;36(1):43–53.
56. Imanshahidi M, Hosseinzadeh H. Pharmacological and therapeutic effects of Berberis vulgaris and its active constituent, berberine. Phytother Res. 2008;22(8):999–1012.
57. Franzblau SG, Cross C. Comparative in vitro antimicrobial activity of Chinese medicinal herbs. J Ethnopharmacol. 1986;15(3):279–288.
58. Singhal KC. Anthelmintic activity of berberine hydrochloride against Syphacia obvelata in mice. Indian J Exp Biol. 1976;14(3):345–347.
59. Romero MR, Efferth T, Serrano MA, et al. Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an ‘in vitro’ replicative system. Antiviral Res. 1986;68(2):75–83.
60. Kuo CL, Chi CW, Liu TY. The anti-inflammatory potential of berberine in vitro and in vivo. Cancer Lett. 1986;203(2):127–137.
61. Ferreira AC, Isomoto H, Moriyama M, et al. Helicobacter and gastric malignancies. Helicobacter. 1986;13 Suppl 1(Suppl 1):28–34.
62. Farinati F, Cardin R, Cassaro M, et al. Helicobacter pylori, inflammation, oxidative damage and gastric cancer: a morphological, biological and molecular pathway. Eur J Cancer Prev. 2008;17(3):195–200.
63. Chang KS. Down-regulation of c-Ki-ras2 gene expression associated with morphologic differentiation in human embryonal carcinoma cells treated with berberine. J Formos Med Assoc. 1991;90(1):10–14.
64. Shen G, Jeong WS, Hu R, et al. Regulation of Nrf2, NF-kappaB, and AP-1 signaling pathways by chemopreventive agents. Antioxid Redox Signal. 2005;7(11-12):1648–1663.
65. Fukuda K, Hibiya Y, Mutoh M. Inhibition of activator protein 1 activity by berberine in human hepatoma cells. Planta Med. 1999;65(4):381–383.
66. Mantena SK, Sharma SD, Katiyar SK. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP. Carcinogenesis. 2006;27(10):2018–2027.
67. Mantena SK, Sharma SD, Katiyar SK. Berberine a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol Cancer Ther. 2006; 5(2):296–308.
68. Letasiová S, Jantová S, Cipák L, et al. Berberine – antiproliferative activity in vitro and induction of apoptosis/necrosis of the U937 and B16 cells. Cancer Lett. 2006;239(2):254–262.
69. Grisendi S, Mecucci C, Falini B, et al. Nucleophosmin and cancer. Nat Rev Cancer. 2006;6(7):493–505.
70. Shay JW, Keith WN. Targeting telomerase for cancer therapeutics. Br J Cancer. 2008;98(4):677–683.
71. Xu Y, Goldkorn A. Telomere and telomerase therapeutics in cancer. Genes (Basel). 2016;7(6):22.
72. Wu HL, Hsu CY, Liu WH, et al. Berberine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of nucleophosmin/B23 and telomerase activity. Int J Cancer. 1999;81(6):923–929.
73. Krey AK, Hahn FE. Berberine: complex with DNA. Science. 1969;66(3906):755–757.
74. Rungsitiyakorn A, Wilairat P, Panijpan B. On the pH dependence of binding of berberine to DNA. J Pharm Pharmacol. 1981;33(2):125–127.
75. Liu F, Wang P, Jiang X, et al. Antisense hypoxia-inducible factor 1alpha gene therapy enhances the therapeutic efficacy of doxorubicin to combat hepatocellular carcinoma. Cancer Sci. 2008;99(10):2055–2061.
76. Chidambara Murthy KN, Jayaprakasha GK, Patil BS. The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells. Eur J Pharmacol. 2012;688(1-3):14–21.
77. Weiner LM, Surana R, Wang S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol. 2010;10(5):317–327.
78. Wang W, Erbe AK, Hank JA, et al. NK Cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy. Front Immunol. 2015;6:368.
79. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12(4):278–87.  
80. Gelderman KA, Tomlinson S, Ross GD, et al. Complement function in mAb-mediated cancer immunotherapy. Trends Immunol. 2004;25(3):158–64.
81. Sharkey RM, Goldenberg DM. Cancer radioimmunotherapy. Immunotherapy. 2011;3(3):349–370.
82. Chen J, Keltner L, Christophersen J, et al. New technology for deep light distribution in tissue for phototherapy. Cancer J. 2002;8(2):154-63.
83. Wust P, Hildebrandt B, Sreenivasa G, et al. Hyperthermia in combined treatment of cancer. Lancet Oncol. 2002;3(8):487–497.
84. Prise KM, O'Sullivan JM. Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer. 2009;9(5):351–360.
85. Chatterjee K, Zhang J, Honbo N, et al. Doxorubicin Cardiomyopathy. Cardiology. 2010;115(2):155–162.
86. Yao X, Panichpisal K, Kurtzman N, et al. Cisplatin Nephrotoxicity: A Review. Am J Med Sci. 2007;334(2):115–124.
87. Voulgaris S, Partheni M, Karamouzis M, et al. Intratumoral doxorubicin in patients with malignant brain gliomas. Am J Clin Oncol. 2002;25(1):60–64.
88. Duvillard C, Romanet P, Cosmidis A, et al. Phase 2 study of intratumoral cisplatin and epinephrine treatment for locally recurrent head and neck tumors. Ann Otol Rhinol Laryngol. 2004;113(3 Pt 1):229–233.
89. Lammers T, Peschke P, Kühnlein R, et al. Effect of Intratumoral Injection on the Biodistribution and the Therapeutic Potential of HPMA Copolymer-Based Drug Delivery Systems. Neoplasia. 2006;8(10):788–795.
90. Medina OP, Zhu Y, Kairemo K. Targeted liposomal drug delivery in cancer. Curr Pharm Des. 2004;10(24):2981–2989.
91. Muzykantov VR. Drug delivery by red blood cells: vascular carriers designed by mother nature. Expert Opin Drug Deliv. 2010;7(4):403–427.
92. Zellmer S, Cevc G. Tumor targeting in vivo by means of thermolabile fusogenic liposomes. J Drug Target. 1996;4(1):19–29.
93. Landesman-Milo D, Ramishetti S, Peer D. Nanomedicine as an emerging platform for metastatic lung cancer therapy. Cancer Metastasis Rev. 2015;34(2):291–301.
94. Peer D, Karp JM, Hong S, et al. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol. 2007;2(12):751–760.
95. Jain A, Jain SK. PEGylation: an approach for drug delivery. A review. Crit Rev Ther Drug Carrier Syst. 2008;25(5):403–447.
96. Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005;10(21):1451–1458.
97. Béduneau A, Saulnier P, Hindré F, et al. Design of targeted lipid nanocapsules by conjugation of whole antibodies and antibody Fab' fragments. Biomaterials. 2007;28(33):4978–4990.
98. Daniels-Wells TR, Penichet ML. Transferrin receptor 1: a target for antibody-mediated cancer therapy. Immunotherapy. 2016;8(9):991–994.
99. Hoffman AS. The origins and evolution of “controlled” drug delivery systems. J Control Release. 2008;132(3):153–163.
100. Lu RM, Chen MS, Chang DK, et al. Targeted drug delivery systems mediated by a novel peptide in breast cancer therapy and imaging. PLoS ONE. 2013;8(6):e66128.
101. Wei Y, Zhou F, Zhang D, et al. A graphene oxide based smart drug delivery system for tumor mitochondria-targeting photodynamic therapy. Nanoscale. 2016;8(6):3530–3538.
102. Noguchi Y, Wu J, Duncan R, et al. Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues. Jpn J Cancer Res. 1998;89(3):307–314.
103. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul. 2001;41:189–207.
104. Burt HM, Zhang X, Toleikis P, et al. Development of copolymers of poly (d, l-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel. Colloids Surf B Biointerfaces. 1999;16(4):161–171.
105. Jin H, Heller DA, Sharma R, et al. Size-dependent cellular uptake and expulsion of single-walled carbon nanotubes: single particle tracking and a generic uptake model for nanoparticles. ACS Nano. 2009;3(1):149–158.
106. Jiang W, Kim BYS, Rutka JT, et al. Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol. 2008;3(3):145–150.
107. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific nanoparticles: Theory to practice. Pharmacol Rev. 2001;53(2):283–318.
108. Walkey CD, Olsen JB, Guo H, et al. Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J Am Chem Soc. 2012;134(4):2139–2147.
109. Lazarovits J, Chen YY, Sykes EA, et al. Nanoparticle-blood interactions: the implications on solid tumour targeting. Chem Commun. 2015;51(14):2756–2767.
110. Hirn S, Semmler-Behnke M, Schleh C, et al. Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration. Eur J Pharm Biopharm. 2011;77(3):407–416.
111. Sonavane G, Tomoda K, Makino K. Biodistribution of colloidal gold nanoparticles after intravenous administration: effect of particle size. Colloids Surf B. 2008;66(2):274–280.
112. Perrault SD, Walkey C, Jennings T, et al. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett. 2009;9(5):1909–1915.
113. Sun C, Lee JS, Zhang M. Magnetic nanoparticles in MR imaging and drug delivery. Adv Drug Deliv Rev. 2008;60(11):1252–1265.
114. Corot C, Robert P, Idee JM, et al. Recent advances in iron oxide nanocrystal technology for medical imaging. Adv Drug Deliv Rev. 2006;58(14):1471–1504.
115. Wang YX, Hussain SM, Krestin GP. Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging. Eur Radiol. 2001;11(11):2319–2331.
116. Hergt R, Dutz S, Müller R, et al. Magnetic particle hyperthermia: nanoparticle magnetism and materials development for cancer therapy. J Phys Condens Matter. 2006;18:S2919–S2934.
117. Glöckl G, Hergt R, Zeisberger M, et al. The effect of field parameters, nanoparticle properties and immobilization on the specific heating power in magnetic particle hyperthermia. J Phys Condens Matter. 2006;8:S2935–S2949.
118. Wanga X, Gub H, Yangc Z. The heating effect of magnetic fluids in an alternating magnetic field. J Magn Magn Mater. 2005;293(1):334–340.
119. Raaphorst GP. Fundamental aspects of hyperthermic biology. In: Field SB and Hand JW. An introduction to the practical aspects of clinical hyperthermia. Taylor and Francis, London; 1990. pp 10–54.
120. Sminia P, van der Zee J, Wondergem J, et al. Effect of hyperthermia on the central nervous system: a review. Int J Hyperthermia. 1994;10(1):1–30.
121. Multhoff G, Botzler C, Wiesnet M, et al. A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer. 1995;61(2):272–279.
122. Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials. 2005;26(18):3995–4021.
123. Ma Y, Manolache S, Denes FS, et al. Plasma synthesis of carbon magnetic nanoparticles and immobilization of doxorubicin for targeted drug delivery. J Biomater Sci Polym Ed. 2004;15(8):1033–1049.
124. Jayakumar OD, Ganguly R, Tyagi AK, et al. Water dispersible Fe3O4 nanoparticles carrying doxorubicin for cancer therapy. J Nanosci Nanotechnol. 2009;9(11):6344–6348.
125. Lubbe AS, Bergemann C, Riess H, et al. Clinical experiences with magnetic drug targeting: a phase I study with 4′-epidoxorubicin in 14 patients with advanced solid tumors. Cancer Res. 1996;56(20):4686–4693.
126. Rudgea SR, Kurtza TL, Vesselya CR, et al. Preparation, characterization, and performance of magnetic iron-carbon composite microparticles for chemotherapy. Biomaterials. 2000;21(14):1411–1420.
127. Takeda S, Mishima F, Fujimoto S, et al. Development of magnetically targeted drug delivery system using superconducting magnet. J Magn Magn Mater. 2007;311(1):367–371.
128. Fernández-Pacheco R, Valdivia JG, Ibarra MR. Magnetic nanoparticles for local drug delivery using magnetic implants. Methods Mol Biol. 2009;544:559–569.
129. Ritter JA, Ebner AD, Daniel KD, et al. Application of high gradient magnetic separation principles to magnetic drug targeting. J Magn Magn Mater. 2004;280(2-3):184–201.
130. Majidi S, Zeinali Sehrig F, Samiei M, et al. Magnetic nanoparticles: Applications in gene delivery and gene therapy. Artif Cells Nanomed Biotechnol. 2016;44(4):1186–1193.
131. Saiyed ZM, Ramchand CN, Telang SD. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support. J Phys Condens Matter. 2008;20(20):204153.
132. Krötz F, de Wit C, Sohn HY, et al. Magnetofection--a highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo. Mol Ther. 2003;7(5 Pt 1):700–710.
133. Mykhaylyka O, Vlaskoua D, Tresilwisedb N, et al. Magnetic nanoparticle formulations for DNA and siRNA delivery. J Magn Magn Mater. 2007;311(1):275–281.
134. Huth S, Lausier J, Gersting SW, et al. Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer. J Gene Med. 2004;6(8):923–936.
135. McBain SC, Griesenbach U, Xenariou S, et al. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays. Nanotechnology. 2008;19(40):405102.
136. Cai D, Mataraza JM, Qin ZH, et al. Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing. Nat Methods. 2005;2(6):449–454.
137. Divakaran SA, Sreekanth KM, Rao KV, et al. D-aminoacid oxidase-Fe2O3 nanoparticle complex mediated antitumor activity in Swiss albino mice. J Cancer Ther. 2011;2:666–674..
138. Bava A, Gornati R, Cappellini F, et al. D-amino acid oxidase-nanoparticle system: a potential novel approach for cancer enzymatic therapy. Nanomedicine (Lond). 2013;8(11):1797–1806.
139. Zhang A, Miao K, Sun H, et al. Tumor heterogeneity reshapes the tumor microenvironment to influence drug resistance. Int J Biol Sci. 2022;18(7):3019–3033.
140. Correia A L, Bissell MJ. The tumor microenvironment is a dominant force in multidrug resistance. Drug Resist Updat. 2012;15(1–2):39–49.
141. Hamm CA, Stevens JW, Xie H, et al. Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors. BMC Cancer. 2010;10:471.
142. Popova NV, Jücker M. The functional role of extracellular matrix proteins in cancer. Cancers(Basel). 2022;14(1):238.
143. Keely PJ. Mechanisms by which the extracellular matrix and integrin signaling act to regulate the switch between tumor suppression and tumor promotion. J Mammary Gland Biol Neoplasia. 2011;16(3):205–219.
144. Levental KR, Yu H, Kass L, et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009;139(5):891–906.
145. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249–257.
146. Hashizume H, Baluk P, Morikawa S, et al. Openings between defective endothelial cells explain tumor vessel leakiness. In Am J Pathol. 2000;156(4):1363–1380.
147. Lamplugh Z, Fan Y. Vascular microenvironment, tumor immunity and immunotherapy. In frontiers in immunology. Frontiers Media SA. 2021.
148. Li Y, Zhao L, Li XF. Hypoxia and the tumor microenvironment. Technol Cancer Res Treat. 2021;20:15330338211036304.
149. Li XF, O’donoghue JA. Hypoxia in microscopic tumors. Cancer Lett. 2008;18;264(2):172–180.
150. Malhotra V, Perry MC. Classical chemotherapy: mechanisms, toxicities and the therapeutic window. Cancer Boil Ther. 2003;2(4 Suppl 1):S2–S4.
151. Gray LH, Conger AD, Eben M, et al. Concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol. 1953;26(312):638–648.
152. Thomlinson RH, Gray LH. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer. 1955;9(4):539–549.
153. Baskar R, Dai J, Wenlong N, et al. Biological response of cancer cells to radiation treatment. Front Mol Biosci. 2014;1:24.
154. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989;49(23):6449–6465.
155. Warburg O. On the origin of cancer cells. Science. 1956;123(3191):309–314.
156. Tannock IF, Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989;49(16):4373–4384.
157. Rankin EB, Giaccia AJ. Hypoxic control of metastasis. Science. 2016;352(6282):175–180.
158. Lin SC, Liao WL, Lee JC, et al. Hypoxia-regulated gene network in drug resistance and cancer progression. Exp Biol Med. 2014;239(7):779–792.
159. Gene E, Semenza GL, Nejfelt MK. Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human. Proc Natl Acad Sci U S A. 1991;88(13):5680–5684.
160. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science. 1998;242(4884):1412–1415.
161. Kapitsinou PP, Haase VH. The VHL tumor suppressor and HIF: Insights from genetic studies in mice. Cell Death Differ. 2008;15(4):650–659.
162. Yu F, White SB, Zhao Q, et al. HIF-1 binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci U S A. 2001;98(17):9630–9635.
163. Lee SH, Jeong D, Han YS, et al. Pivotal role of vascular endothelial growth factor pathway in tumor angiogenesis. Ann Surg Treat Res. 2015;89(1):1–8.
164. Cao D, Hou M, Guan YS, et al. Expression of HIF-1alpha and VEGF in colorectal cancer: Association with clinical outcomes and prognostic implications. BMC Cancer. 2009;9:432.
165. Franovic A, Gunaratnam L, Smith K, et al. Translational up-regulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer. Proc Natl Acad Sci U S A. 2007;104(32):13092–13097.  
166. Rauth AM, Mohindra JK, Tannock IF. Activity of mitomycin C for aerobic and hypoxic cells in vitro and in vivo. Cancer Res. 1983;43(9):4154–4158.
167. Hall EJ, Roizin-Towle L. Hypoxic sensitizers: Radiobiological studies at the cellular level. Radiology. 1975;117(2):453–457.
168. Mohindra JK, Rauth AM. Increased cell killing by metronidazole and nitrofurazone of hypoxic compared to aerobic mammalian cells. Cancer Res. 1976;36(3):930–936.
169. Moore BA, Palcic B, Skarsgard LD. Radiosensitizing and toxic effects of the 2-nitroimidazole Ro-07-0582 in hypoxic mammalian cells. Radiat Res. 1976;67(3):459–473.
170. Lin AJ, Cosby LA, Shansky CW, et al. Bioreductive alkylating agents: 1. Benzoquinone derivatives. J Med Chem. 1972;15(12):1247–1252.
171. Zeman EM, Brown JM, Lemmon MJ, et al. SR-4233: a new agent with high selective toxicity for hypoxic mammalian cells. Int J Radiat Oncol Biol Phys. 1986;12(7):1239–1242.
172. Wilson WR. Tumour hypoxia: challenges for cancer chemotherapy. In: Waring M J, Ponder BAJ. Cancer Biology and Medicine. Lancaster: Kluwer Academic Publishers, 1992;3:87–131.
173. Ortiz de Montellano PR. Cytochrome P450-activated prodrugs. Future Med Chem. 2013;5(2):213–228.
174. Brown JM. SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours. Br J Cancer. 1993;67(6):1163–1170.
175. Murphy MP. How mitochondria produce reactive oxygen species. Biochem J. 2009;417(1):1–13.
176. Lee AE, Wilson WR. Hypoxia-dependent retinal toxicity of bioreductive anticancer prodrugs in mice. Toxicol Appl Pharmacol. 2000;163(1):50–59.
177. Dorie MJ, Brown JM. Tumor-specific, schedule dependent interaction between tirapazamine (SR 4233) and cisplatin. Cancer Res. 1993;53(19):4633–4636.
178. Kovacs MS, Hocking DJ, Evans JW, et al. Cisplatin anti-tumour potentiation by tirapazamine results from a hypoxia-dependent cellular sensitization to cisplatin. Br J Cancer. 1999;80(8):1245–1251.
179. Daniels JS, Gates KS. DNA cleavage by the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (SR4233): Evidence for involvement of hydroxyl radical. J Am Chem Soc. 1996;118(14):3380–3385.
180. Zagorevskii D, Song M, Breneman C, et al. A mass spectrometry study of tirapazamine and its metabolites. Insights into the mechanism of metabolic transformations and the characterization of reaction intermediates. J Am Soc Mass Spectrom. 2003;14(8):881–892.
181. Anderson RF, Shinde SS, Hay MP, et al. Activation of 3-amino-1,2,4-benzotriazine 1,4-dioxide antitumor agents to oxidizing species following their one-electron reduction. J Am Chem Soc. 2003;125(3):748–756.
182. Patterson LH, Murray GI. Tumour cytochrome P450 and drug activation. Curr Pharm Des. 2002;8(15):1335–1347.
183. Patterson LH. Bio-reductively activated anti-tumor N-oxides: the case of AQ4N, a unique approach to hypoxia activated cancer chemotherapy. Drug Metab Rev. 2002;34(3):581–592.
184. Greco O, Dachs GU. Gene directed enzyme/pro-drug therapy of cancer: historical appraisal and future prospectives. J Cell Physiol. 2001;187(1):22–36.
185. Binley K, Askham Z, Martin L, et al. Hypoxia-mediated tumour targeting. Gene Ther. 2003;10(7):540–549.
186. Trinh QT, Austin EA, Murray DM, et al. Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. Cancer Res. 1995;55(21):4808–4812.
187. McCarthy HO, Yakkundi A, McErlane V, et al. Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N. Cancer Gene Ther. 2003;10(1):40–48.
188. Griffiths L, Binley K, Iqball S, et al. The macrophage - a novel system to deliver gene therapy to pathological hypoxia. Gene Ther. 2000;7(3):255–262.
189. Burke B, Tang N, Corke KP, et al. Expression of HIF-1alpha by human macrophages: implications for the use of macrophages in hypoxia-regulated cancer gene therapy. J Pathol. 2002;196(2):204–212.
190. Kung AL, Wang S, Klco JM, et al. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nature Med. 2000;6(12):1335–1340.
191. Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1alpha protein via the proteosome pathway in prostate cancer cells. Cancer Res. 2002;62(9):2478–2482.
192. Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-1α enhances the therapeutic efficacy of cancer immunotherapy. Gene Ther. 2001;8(8):638–645.  
193. Ai Z, Lu Y, Qiu S, et al. Overcoming cisplatin resistance of ovarian cancer cells by targeting HIF-1-regulated cancer metabolism. Cancer Lett. 2016;373(1):36–44.
194. Masoud GN, Li W. HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B. 2015;5(5):378–389.
195. Jung HY, Fattet L, Yang J. Molecular pathways: linking tumor microenvironment to epithelial-mesenchymal transition in metastasis. Clin Cancer Res. 2015;21(5):962–968.
196. Zhao T, Ren H, Jia L, et al. Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma. Oncotarget. 2015;6(4):2250–2262.
197. Nowotny A. Antitumor effects of endotoxins. In: Berry LJ. Handbook of Endotoxin. Elsevier Science, Amsterdam. 1985;3:389–448.
198. Lemmon MJ, Elwell JH, Brehm JK, et al. Anaerobic bacteria as a gene delivery system to tumors. Proc Am Assoc Cancer Res. 1994;35:374.
199. Lemmon MJ, van Zijl P, Fox ME, et al. Anaerobic bacteria as a gene delivery system that is controlled by the tumour microenvironment. Gene Ther. 1997;4(8):791–796.
200. Jain KK. Use of bacteria as anticancer agents. Expert Opin Biol Ther. 2001;1(2):291–300.
201. Fox ME, Lemmon MJ, Mauchline ML, et al. Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther. 1996;3(2):173–178
202. Gardlik R, Behuliak M, Palffy R, et al. Gene therapy for cancer: bacteria-mediated anti-angiogenesis therapy. Gene Ther. 2011;18(5):425–431.
203. Hu CM, Aryal S, Zhang L. Nanoparticle-assisted combination therapies for effective cancer treatment. Ther Deliv. 2010;1(2):323–334.
204. Bai M, Shen M, Teng Y, et al. Enhanced therapeutic effect of Adriamycin on multidrug resistant breast cancer by the ABCG2-siRNA loaded polymeric nanoparticles assisted with ultrasound. Oncotarget. 2015;6(41):43779–43790.
205. Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-1α enhances the therapeutic efficacy of cancer immunotherapy. Gene Ther. 2001;8(8):638–645.
206. Nair GG, Nair CKK. Sanazole directed targeting of silver nanoparticle drug complex to tumor mass: a preclinical investigation in murine model. J Cancer Res Ther. 2014;10(4):979–984.
207. Sreeja S, Nair CKK. Chemo-directed specific targeting of nanoparticle-doxorubicin complexes to tumor in animal model. J Drug Deliv Sci Technol. 2016;31:167–175.  
208. Sreeja S, Nair CKK. Tumor control by hypoxia-specific chemotargeting of iron-oxide nanoparticle - Berberine complexes in a mouse model. Life Sci. 2018;195:71–80.
209. Ellerby HM, Bredesen DE, Fujimura S, et al. Hunter-killer peptide (HKP) for targeted therapy. J Med Chem. 2018;51(19):5887–5892.