Prostate cancer is still the most common cancer diagnosed in American men after skin cancer. According to the American Cancer Society, there will be approximately 164,690 new cases and 29,430 deaths from prostate cancer for 2018. The increased difficulty in the treatment of metastasized advanced prostate cancer raises the need for the exploration of novel treatments. This study investigated the augmentation of very low dose radiation (VLDR, 20mGy/hr) on the treatment efficacy of genistein (Gn) in DU-145 androgen-independent prostate cancer cells. The objective was to assess the efficacy of the combinative treatment regimen relative to the single treatments. Our data revealed that exposing DU-145 cells to VLDR for 20 minutes before treatment with (Gn), significantly increased the therapeutic efficiency of genistein (Gn); the combination treatment (VLDR-Gn) caused significantly more apoptotic cell death in DU-145 cells. The present data (from DU-145 cells) is in conformity with results of previous studies (on LNCaP and PC3 cells); indicating that both androgen-dependent and androgen-independent prostate cancer cells are sensitive to (VLDR-Gn) combinative regimen. This observation provides a rationale and justification for further investigation in the field of radiation-phytochemical combinative treatment.

Keywords: Prostate cancer, Very Low Dose Radiation (VLDR), Genistein (Gn), DU-145, Apoptosis, Pyroelectricity

Cancer is a complex disease that occurs as a result of accumulated mutations, genetic instability, and altered cellular metabolism, contributing to malignant transformation and to the initiation, growth, and maintenance/proliferation of tumors.¹ Prostate cancer (PCa) is still the most common non-skin cancer among men in the United States.² The American Cancer Society has estimated 164,690 new cases and 29,430 deaths from prostate cancer for 2018.³ The standard treatment modalities (surgery, radio-chemotherapy, hormonal therapy) have been effective in improving the lifestyle of patients. Although the locally confined disease is treatable, treatment of the metastasized prostate cancer is still incurable with mostly guarded prognosis.^4,5 The development of resistance to both radiation and chemotherapy has limited the efficacy of current therapeutic interventions for PCa. This has necessitated the search for novel and safer alternative therapeutic regimens. One way to circumvent potential treatment-induced resistance is combination treatment, using two or more therapies that may be mutually inclusive or exclusive in terms of their mechanism of actions. There has been a focus on phytotherapy, exploring the micronutrients in plant and plant products; and the phytochemical Genistein isoflavone has been in the limelight. Genistein (4, 5, 7-trihydroxyisoflavone) is derived from soy and soy products. Increased plasma levels of isoflavone have been inversely correlated with PCa risk factors. In this study, we explored the therapeutic efficacy of genistein isoflavone (Gn) in combination with a very low dose of radiation (VLDR), in DU-145 hormone-independent prostate cancer cells.

Genistein isoflavone

Genistein isoflavone (Gn) (4′, 5′, 7-trihydroxyisoflavone) is a major dietary phytochemical in soy that has shown therapeutic potential against various human cancer cells in vitro and in animal models.^6–8 The anti-cancer effects of (Gn) has been demonstrated in breast, lung, prostate, lymphoma, leukemia, and head and neck cancers.^6,9–20 The specific mechanisms of (Gn) action on cancer cells have been the focus of ongoing investigations for years. Genistein is structurally related to estrogen and can, therefore, exhibit both estrogenic and anti-estrogenic activities.21 It has the ability to bind estrogenic receptors and regulate cell growth and proliferation in both androgen-dependent and androgen-independent cancer cells.13^,22 In addition, (Gn) exhibits several pharmacological effects unrelated to its estrogenic properties- including: inhibition of angiogenesis and microsomal lipid peroxidation,^21–24 inhibition of topoisomerase I & II,6 inhibition of telomerase activity,²⁵ inhibition of tyrosine kinase signaling pathway, inhibition of two cell survival proteins - Akt and NF-kβ;²² two anti-apoptotic proteins - Bcl-2 and Bcl-XL^16,20,26 and upregulation of the pro-apoptotic proteins - Bax and Bad9^{,15–17, 27,28} and a decrease in vascular endothelial growth factor (VEGF) correlating with a reduction in angiogenesis¹⁶ have also been reported. At low concentrations, we have been able to demonstrate that (Gn) act as an antioxidant and at high concentration, it operates as a pro-oxidant. High concentrations of (Gn) create an increase in reactive oxygen species (ROS) production, which potentially leads to DNA damage and cell arrest at the G2/M cell cycle phase.^21,29 There is further evidence that the mechanistic pathways through which (Gn) inhibits cancer cell growth and proliferation include: altering signal transduction pathways, cell cycle regulatory molecules, and activation of the caspase proteases.^13,30 Another study shows the ability of genistein (Gn) to block facilitative glucose transporter 1- GLUT1 activity when it acts as a tyrosine kinase inhibitor, thereby decreasing the affinity of d-glucose for its external binding site on the cell membrane.³¹ Hence, it has the potential to inhibit glycolytic metabolism in cancer cells. It is, therefore, possible that one of the mechanisms through which (Gn) induces apoptosis in cancer cells is the modulation of protein, enzymes, and biomarkers associated with angiogenesis, metabolism and tumor growth and invasion. Genistein has significantly fewer side effects and less cytotoxicity compared to standard chemo-radiotherapy. Genistein in combination with very low dose radiation (VLDR) therapy could potentially offer a safer clinical option.

Very Low Dose Radiation therapy (VLDR) and Hyper-Radiosensitivity (HRS)

Radiotherapy plays a pivotal role in standard cancer therapeutic regimen, mostly in combination with chemotherapy. The BEIR VII report defined low doses of radiation as those that do not exceed 100 mGy.^32–34 Dual responses have been described in most mammalian cells at low doses of ionizing radiation.^35,36 The first response prevents and/or repairs DNA damage to keep cells viable and functioning properly. The second response removes damaged cells by inducing apoptosis, terminal differentiation, and immune responses; thus eliminating mutated cells and reducing genomic instability in exposed cells/tissues.^37,38 Unlike the high-power sources of X-ray radiations-linear accelerators and synchrotrons used for radiotherapy,³⁹ recent investigations have been focused on the development of novel and cheaper low-power milli/micro/nano X-ray sources for low dose radiation.^40,41

The goals of combined treatment protocols are to improve the local control and to prevent distant micro-metastases, which may be flawed by the intrinsic radio-resistant and chemo-resistant properties of most solid tumors.⁴² Studies have suggested that many cancer cells are sensitive to low doses or fractionated doses of radiation, and that (very) low doses of radiation can be used to enhance or potentiate the chemotherapeutic effect of most drugs, while at the same time decreasing the development of tumor resistance.^43–46 At low doses (LD) or very low doses (VLD) of radiation, hyper-radiosensitivity (HRS) has been demonstrated to be responsible for the increased sensitivity observed in these cells at doses less than 0.1 Gy.^47,48 Our hypothesis is that the use of very low dose radiation (VLDR) in combination with genistein isoflavone (Gn) will inhibit treatment-induced resistance and induce apoptotic cell death in DU-145 PCa cells at a faster rate and with significantly lower cytotoxicity.

Cell line: DU-145 androgen-independent prostate cancer cell line was purchased from the American Type Culture Collection (Manassas, VA). The cells were stored at -90 ⁰C until used. 

Reagents: MTT reagent and RPMI 1640 culture medium & Fetal Bovine Serum (FBS) were purchased from Fisher Scientific (Waltham, MA); and Genistein (4’, 5’, 7-trihydroxyisoflaven) from Indoline Chemical Co. (Summerville, NJ). Genistein was constituted in DMSO (Dimethyl Sulfoxide) to obtain a stock solution of Gn-500μM. The stock solution was diluted with RPMI 1640 culture media without FBS to varying working concentrations of Gn (10µM, 20µM, 30µM, 40µM, 50µM, 60µM, and 80µM) (Gn10 - Gn80). These were stored at 4 ⁰C until used. 

Cell culture: DU-145 cells were thawed and centrifuged at 3000rpm. The pelleted cells were resuspended in RPMI 1640 culture medium with FBS, transferred into 25 cm² culture flasks and cultured-grown/incubated at 37 ⁰C and 5% CO₂ for 36 to 48 hrs to achieve 80-90% confluence. Next, adherent cells were scraped with Pasteur pipette or trypsinized into monolayer suspensions. Culture medium was added and cell concentration in the flasks was determined with a hemocytometer. To culture the cells in 96-well microtiter plates (MTTP), 100μl of the suspension containing approximately 1x 10⁴ cells were transferred into each well in the MTTPs. The plates were cultured/incubated at 37 ⁰C and 5% CO₂ for 24 to 48 hrs to achieve 80-90% confluence; after which the MTTPs were grouped into A-MTTPs (for radiation) and B-MTTPs (non-radiation) to be treated with and/or without (Gn). 

In vitro pyroelectric X-ray irradiation

DU-145 cells were cultured as previously described above. Cells in A-MTTPs were irradiated with a portable pyroelectric X-ray generator (Cool X, AMPTEX INC, Bedford, MA). Cells were exposed to a single dose of very low dose radiation (VLDR) of 20mGy/hour for 20 minutes. The risk and exposure to the lab were considered and a custom lead box was designed to prevent any long-term and repeated X-ray exposure in the lab. The appropriate condition of 37 ⁰C and 5% CO₂ was maintained within the X-ray box through a BD GasPak EZ Anaerobe Container system.

MTT Assay of irradiated and non-irradiated treated DU-145 cells

MTT (3- [4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) was utilized to assess the phytotherapeutic efficacy of genistein (Gn) on irradiated (A-MTTPs) and non-irradiated (B-MTTPs) hormone independent DU-145 cells in single and combination treatments. The DU-145 cells were cultured and treated as previously described above, and the plates were treated with 100μl of the constituted doses of (Gn10-80) as follows: i) A-MTTPs (no Gn), ii) B-MTTPs + Gn10-80 (single treatment) and, iii) A-MTTPs + Gn10-80 (combination treatment). All the plates (MTTPs) were incubated at 37 ⁰C and 5% CO₂ and subjected to the MTT assay at 6 hrs, 24 hrs, and 48 hrs post-treatment. The assay was performed according to the manufacturer’s instructions from Fisher Scientific (Waltham, MA). The absorbance was read at 570nm in the Multiskan biochromatic automated microplate reader (Multiskan, DC). 

The post-treatment radio-phytochemical effect on the DU-145 cells was assessed by the following determinants/equations:

I) Percentage Cell Survival Rate (%CSR) was expressed as:

                %CSR= [(AT – AB) / (AC – AB)] x 100

Where At= Absorbance at treated, AB= Absorbance at Blank, AC= Absorbance at control.

II) Genistein Enhancement ratio (GER) was measured by obtaining the %CSR for the Genistein alone and the %CSR of the VLDR and Genistein combination as follows:

                GER = [%CRS (Gn)] / [%CSR (VLDR +Gn)]

III) Differential Growth Inhibition Rate (DGIR) between Genistein alone vs. VLDR enhanced

Genistein combinative treatment can be expressed as:

                DGIR= [%CRS (Gn)] - [%CSR (VLDR +Gn)]

Fluorescence microscopy Study

This was to ascertain the mode of treatment-induced cell death in the DU-145 prostate cancer cells. A cocktail of two dyes (Acridine Orange and Ethidium Bromide) with different emission spectra were constituted and used to study the color differentials of the treated cells. With this specific stain, cells with light to intense orange fluorescent represent different stages of apoptosis, dark orange being the latest stage. Green would represent viable cells and red fluorescent cells would represent necrotic cell death. Briefly, DU-145 cells were initially cultured and treated as previously described in materials and methods. For the fluorescence microscope study, 50μl of each suspension was discharged onto microscope slides and 10μl of the dye cocktail was added and thoroughly mixed. Slides were covered lightly with coverslips and examined under a fluorescence microscope at 100x magnification. From each slide, four optical views were examined for cells and color differentials; and the number of each type/group of colored cells and the overall percent apoptosis was estimated. For each of the treatment groups, fluorescence micro photographed pictures were taken directly from under the fluorescence microscope with a Samsung Galaxy S9+ camera (Samsung: Galaxy S9 February 2018, Seoul, South Korea).

Statistical analysis

Student t-test was performed and P-value ≤ 0.05 was considered significant.

Results

We investigated the effects of genistein isoflavone (Gn) and very low dose radiation (VLDR) on androgen resistant/independent DU-145 (AR-, P53-) prostate cancer cells. The results are summarized in Figures 1–4 and Tables 1 & 2.

Figure 1 The % CSR was assessed using MTT assay in DU-145 prostate cancer. (%CSR: y-axis) was plotted against the Control (no treatment), VLDR alone, Gn 40μM only, and combinative VLDR and Gn 40μM. A significant difference in the % CSR between control and a single dose of VLDR of 21.43% was found at the significance of (p< 0.02). Also, a significant difference of 16.87% was discovered with (p<0.006) between Gn 40μM alone and the combination of Gn 40μM and VLDR. The results were means+STD of three experiments performed in triplicates.

Figure 2a The %CSR was assessed with the MTT assay on DU-145 prostate cancer cells at 6 hrs post-treatment. The (%CSR: y-axis) was plotted against (Gn40-80) and (VLDR+Gn40-80) (x-axis). The relative differences in (%CSR) were compared. Dose dependent significant decrease (p<0.05) was observed. The results were means + STD of three experiments performed in triplicates.

Figure 2b The %CSR was assessed with the MTT assay on DU-145 prostate cancer cells at 48 hrs post-treatment. The (%CSR: y-axis) was plotted against (Gn40-80) and (VLDR+Gn40-80) (x-axis). The relative differences in (%CSR) were compared. Dose dependent significant decrease (p<0.05) was observed. The results were means+STD of three experiments performed in triplicates.

Figure 3 Photomicrographs (100x) of DU-145 cells treated with or without VLDR and/or Genistein. (A) Du-145 cell line at culture day 0. (B) DU-145 cells at culture 48 hours prior treatment. (C) 24 hrs post VLDR treatment. (D) 24 hrs post VLDR + Gn 50μM combinative treatment.

Figure 4 The morphological observation of the cell death type with Acridine Orange/ Ethidium Bromide (AO/EB) staining. DU-145 prostate cancer cells (A) were untreated (control), (B) were treated with single treatment of VLDR only (C) were treated with a single dose of Gn 60μΜ only, (D) were treated with a single treatment of VLDR and a single dose of Gn 60μΜ.

Figure 1 shows that VLDR significantly decreased the percent cell survival rate post-irradiation. There was a 21.43% decrease in DU-145 survival rate compared with the control (non-irradiated) DU-145 cells. The difference was significant (p<0.02). Also, a significant difference of 16.87% was discovered with (p<0.006) between Gn 40μM alone and combination of Gn 40μM and VLDR. 

VLDR enhances the effects of genistein on DU-145 (AR-, P53-) cell line

A single dose of VLDR enhanced the inhibitory effects of Genistein on the androgen-independent DU-145 cells (Tables 1 & 2 and Figure 2). At 6 hr. post-treatment, as summarized in Table 1, there was a significant decrease in the %CSR in various ratios in different doses. The Differential Growth Inhibition Rate (DGIR) was 17.27 (p< 0.04), 17.98 (p<0.02) and 17.97 (p<0.01) in Gn 40μM, Gn 60μM, and Gn 80μM respectively; the Genistein Enhancement Ratio (GER) were 1.35, 1.52, 2.24 in Gn 40μM, Gn 60μM, and Gn80μM respectively with significant differences (Table 1). At 48 hr. post-treatment, as shown in Table 2, there was a significant decrease in the % CSR in the various ratios in different doses, and the DGIR in Gn 40μM, Gn 60μM, and Gn 80μM were 16.87, 12.8, and 7.76 respectively, with significant differences (Table 2). The overall results revealed a dose and time-dependency of the Gn and VLDR in single and combination on the DU-145 cells; with significant differences in and between the parameters (Tables 1 and 2). 

Microscopic analysis of DU-145 cells

Inverted microscopy examination:

The DU-145 cells were grown and treated as previously described under materials and methods. The cells were examined under the microscope at 100x magnification. Figure 3 shows the dramatic inhibitory effect of irradiation (VLDR) and (Gn-VLDR) combination on the cells with a significant difference (P <0.05) between the treatment groups. Growth inhibition was dose and time-dependent. The highest growth inhibition and cell death were seen in the combination treatment at 24-hr post-treatment (Figure 3D). These microscopic observations correlate significantly with data/results in Tables 1 and 2.

Fluorescence microscopy studies

Fluorescence microscopy was studied to determine treatment-induced apoptotic cell death. The DU-145 cells were treated and cultured as previously written. Fluorescence microscopy stains and staining procedures were also discussed previously. Cells were examined under four optical views at 100x on each slide, and the percentage of each type of cells (live, apoptotic, necrotic) was roughly estimated. The fluorescence microphotographs are shown in Figures 4A-4D.       

Light microscopy/ELISA

In this study, we demonstrated the effects of priming DU-145 human prostate cancer cell line with very low dose radiation (VLDR) of 20mGy/hours approximately (0.333mGy/min) prior to chemo treatment with (Gn). The data revealed a significantly increased sensitivity of the cells to genistein isoflavone when primed with VLDR, in a dose and time-dependent manner. The combination treatment with (VLDR-Gn) exerted most significant growth inhibitory effect on the hormone-independent DU-145 cells. These results are consistent with the results of previously done studies on two different prostate cancer cells - PC3 and LNCaP.⁴⁵ This is the first irradiation-phytochemical study on DU-145 cells, to the best of our knowledge. The EC50 of the (VLDR-Gn) combination was significantly less than the EC50 of the single treatment with VLDR or Gn; implying greater efficacy of the combination treatment and augmentation of genistein’s action on DU-145 cells by irradiation with VLDR. This observation is also in conformity with that observed in PC3 (hormone-independent) and LNCaP (hormone-dependent) prostate cancer cells in previous studies.⁴⁵ However, the results indicated that DU-145 cells are less sensitive to the Gn and (VLDR-Gn) combination at lower doses of Gn 10μM. It is critical to note that the results/observation of the microscopy studies (inverted and fluorescence) correlated significantly with the results of the MTT/-ELISA assays, indicating the consistency/validity and accuracy of the results.

Fluorescence microscopy

The mode/type of treatment-induced cell death in the DU-145 cells by the different treatment regimens is shown in Figures 4A through 4D. The fluorescence microscopy revealed that the treatment-induced growth inhibition of the DU-145 cells was mostly due to apoptotic cell death. The (VLDR-Gn) combination induced significantly higher apoptosis in the cells than the VLDR or Gn alone. The % apoptosis at the EC50 of the (VLDR-Gn) combination is significantly higher than the % apoptosis at the EC50 of VLDR or Gn alone; implying significantly less cytotoxicity by the (VLDR-Gn) combination, and significant potential clinical implication. This present result is similar to that found in previous reports on LNCaP and PC3 cells.⁴⁵ The same trend of apoptosis induction was seen. It is crucial to note that irradiation of the cells prior to treatment with genistein lowered the dose of (Gn) with increasing efficacy of apoptosis induction in the cells. This report supports the fact that combining chemotherapy with radiation treatment could offer significant clinical benefits by decreasing cytotoxicity and subsequent side effects in patients.^45,49,50 Preliminary data from limited studies in our lab revealed that (VLDR-Gn) combination inhibited treatment-induced resistance to apoptosis in the prostate cancer cells. Further studies are ongoing.

Our study demonstrated that hormone-independent DU-145 cells became more sensitive to genistein when primed with VLDR for 20 min prior to treatment in a dose-dependent manner than when treated with (Gn) alone. The combination treatment induced significantly higher/more apoptotic cell death in the DU-145 cells at lower (Gn) concentration; implying that the (VLDR-Gn) combination treatment would be less cytotoxic. If this observation can be demonstrated in in vivo studies, the outcome will be clinically significant. Further studies are ongoing. This is the first study to the best of our knowledge to investigate the effects of VLDR on the DU-145 cells sensitivity to genistein isoflavone in vitro.

Author declares that there is no conflicts of interest.

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