J Cancer Prev 2022; 27(3): 170-181
Published online September 30, 2022
© Korean Society of Cancer Prevention
1Cancer Biology Laboratory, School of Life Sciences, 2Special Centre for Systems Medicine, Jawaharlal Nehru University, New Delhi, India
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Emergence of radioresistance in prostate cancer (PCa) cells is a major obstacle in cancer therapy and contributes to the relapse of the disease. EGF receptor (EGFR) signaling plays an important role in the development of radioresistance. Herein, we have assessed the modulatory effects of silibinin on radiation-induced resistance via DNA repair pathways in EGFR-knockdown DU145 cells. shRNA-based silencing of EGFR was done in radioresistant human PCa DU145 cells and effects of ionizing radiation (IR) and silibinin were assessed using clonogenic and trypan blue assays. Furthermore, radiosensitizing effects of silibinin on PCa in context with EGFR were analyzed using flow cytometry, comet assay, and immunoblotting. Silibinin decreased the colony formation ability with an increased death of DU145 cells exposed to IR (5 Gray), with a concomitant decrease in Rad51 protein expression. Silibinin (25 μM) augmented the IR-induced cytotoxic effect in EGFR-knockdown PCa cells, along with induction of G2/M phase cell cycle arrest. Further, we studied homologous recombination (HR) and non-homologous end joining (NHEJ) pathways in silibinin-induced DNA double-strand breaks in EGFR-knockdown DU145 cells. Silibinin down-regulated the expression of Rad51 and DNA-dependent protein kinase proteins without any considerable effect on Ku70 and Ku80 in IR-exposed EGFR-knockdown PCa cells. The pro-survival signaling proteins, phospho-extracellular signal-regulated kinases (ERK)1/2, phospho-Akt and phospho-STAT3 were decreased by silibinin in EGFR-deficient PCa cells. These findings suggest a novel mechanism of silibinin-induced radiosensitization of PCa cells by targeting DNA repair pathways, HR and NHEJ, and suppressing the pro-survival signaling pathways, ERK1/2, Akt and STAT3, in EGFR-knockdown PCa cells.
Keywords: Prostatic neoplasms, Silybin, Radiosensitization, Epidermal growth factor receptor, DNA repair pathways
Prostate cancer (PCa) usually originates from epithelial cells in prostate gland, a major exocrine gland having functional association with reproduction in men. PCa is the second most commonly diagnosed cancer in men after the lung cancer with higher prevalence in developed countries. Since the incidence of PCa is rising [1,2], the improvement in its management including active surveillance, chemotherapy and radiation therapy (RT) is desired . Over the years, RT is pivotal in management of PCa with curative and palliative intent [4-6]. However, development of therapeutic resistance to the RT is still one of the major stumbles while treating PCa patients.
Emergence of therapeutic resistance to RT in tumor cells is a complex process which is regulated by several factors including DNA damage and repair, mutations, chromosomal instability, activation of signaling pathways to adapt to radiotherapy-induced changes leading to development of radioresistance . Ionizing radiation (IR)-induced DNA strand breaks can trigger a cascade of signaling activation resulting in DNA damage responses (DDRs) which rescue the cancer cells from radiation injuries via inducing DNA repair activation and cell cycle arrest . The homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways mediate the activation of double strand breaks (DSBs) repair genes and were found to be altered and associated with development of radioresistance in cancer . Studies have also highlighted the importance of targeting NHEJ and HR pathways in radiosensitizing the tumor cells . Therefore, the understanding of mechanistic insights of regulation of these major DNA repair pathways in PCa is warranted.
Studies have suggested the role of membrane receptor tyrosine kinase, EGF receptor (EGFR), in regulating DNA repair machinery through HR and NHEJ pathways. Recently, we reported the key role of EGFR in regulation of intrinsic radioresistance in PCa by targeting DNA DSBs repair proteins, DNA-dependent protein kinase (PK) and Rad51 . Some plant-derived small molecules have gained significant attention due to their lower toxicity to normal tissues and capability to sensitize the cancer cells to the radiation [12-14]. Our previous study has shown the role of small molecule, silibinin in radiosensitizing PCa cells by inhibiting the radiation-induced nuclear translocation of EGFR .
The mechanistic insights of silibinin-mediated radiosensitizing effect by regulating major DNA DSBs repair pathways, HR and NHEJ, through EGFR signaling have not been studied in PCa. In the present study, we investigated the effect of silibinin on the IR-induced biological and molecular events in EGFR-knockdown radioresistant PCa cells. Our findings suggested Rad51 and DNA-PK as molecular targets of silibinin in radiosensitizing the EGFR-deficient PCa cells by attenuating HR and NHEJ pathways.
Antibodies for Rad51, Ku70, Ku80, p53, Akt, extracellular signal-regulated kinase (ERK)1/2, STAT3, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 1 (CDK1), Cdc25c, and phosphoproteins (p-CDK1-Tyr15, p-p53-Ser15, p-Akt-Ser473, p-ERK1/2-Thr202/Tyr204, p-STAT3-Tyr705) were purchased from Cell Signaling Technology (Beverly, MA, USA). DNA-PK and cyclin B1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin antibody was from Sigma Aldrich (St. Louis, MO, USA). Silibinin (S0417) was purchased from Sigma Aldrich.
Prostate carcinoma DU145 cells (NCCS, Pune, India) were cultured in RPMI-1640 media supplemented with 10% FBS (Gibco, Mumbai, India) and 1× Penicillin-Streptomycin-Amphotericin antibiotic. Cell cultures were maintained in 5% CO2 humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C temperature.
The recptor tyrosine kinase, EGFR, was knockdown in radioresistant DU145 PCa cells using lentiviral-based system and the cloning vector, pLKO.1. Experimentally validated shEGFR sequence for knockdown of the kinase was retrieved from https://www.sigmaaldrich.com/IN/en/semi-configurators/shrna?activeLink=selectClones and cloned in lentiviral vector, pLKO.1. Further, lentiviral particles were generated with the help of helper plasmids (psPAX2 and pMD2.G) and particles were transduced to generate stable EGFR-knockdown DU145 cells as detailed in the previous study .
Briefly, DU145 vector control (pLKO.1) and EGFR knockdown cells (shEGFR) were harvested and seeded at an appropriate density in a 6-well plate. After 24 hours, treatments of silibinin (25 µM) and/or IR (5 Gray [Gy]) were given and cells were maintained at 37°C in an incubator for 8 days. At the end of the experiment, cells were fixed with 4% formaldehyde and stained with 0.05% crystal violet as described in the previous study , and colonies containing more than 50 cells were counted.
DU145 vector control (pLKO.1) and EGFR knockdown cells (shEGFR) were used for the trypan blue dye exclusion assay. In brief, after trypsinization, cells were counted and seeded at a density of 4×104 cells/well in a 12-well plate and treated with silibinin (25 µM) and/or IR (5 Gy). After the 48-hour treatments, cells were trypsinized, collected and processed for trypan blue staining and counted for live and dead cells as described earlier . The coefficient of drug interaction (CDI) value was calculated as detailed in earlier studies [18,19]. The CDI value < 1 and > 1 indicates synergistic and antagonistic effects, respectively, while CDI value = 1 means the effect in combination is additive [18,19].
Briefly, DU145 vector control (pLKO.1) and EGFR knockdown cells (shEGFR) were seeded at a density of 4×104 cells/well and after 24 hours, treated with silibinin (25 µM) and/or IR (5 Gy). After the 48-hour treatment, cells were harvested and resuspended in FACS cocktail solution [RNaseA (10 µg/mL), propidium iodide (25 µg/mL), saponin (0.2%) and Ethylenediaminetetraacetic acid (0.1 mM)] and kept overnight in dark at 4°C . The cells were analyzed for cell cycle phase distribution by FACS (BD FACSAria, Franklin Lakes, NJ, USA) and data was analyzed using FACS Diva Software (Becton Dickinson, Franklin Lakes, NJ, USA).
DNA strand breaks were analyzed using the alkaline comet assay as described earlier . Briefly, cells were trypsinized, counted and seeded at a density of 4×104 cells/well in 12-well plate and after 24 hours, treated with silibinin (25 µM) and/or IR (5 Gy). At the end of the treatments, cells were trypsinized briefly and 15 × 103 cells/0.5 mL in low melting point agarose was coated on the surface of a microscopic slide. Slides were then processed in single cell gel electrophoresis and stained with ethidium bromide (4 µg/mL) and subsequently visualized in fluorescent microscopy for the images. CometScore 2.0 software (http://rexhoover.com/index.php?id=cometscore) was used for scoring comet tail length and tail DNA percentage .
Cells were seeded and treated with silibinin and IR as desired. After respective treatment time points, cells were harvested and whole cell lysate preparation, protein quantification and immunoblotting were performed as detailed in the earlier study .
Cells were irradiated at 5 Gy dose in 1× PBS followed by treatment with low dose of silibinin (25 µM). Cells were irradiated in common instrument fac60Co gamma chamber (Model 5000A; Bhabha Atomic Research Centre, Mumbai, India) at a dose rate 0.5 Gy/second.
All data were statistically analyzed using GraphPad Prism (version 5.0.0; GraphPad Software, San Diego, CA, USA). The analysis for comet assay data was done using CometScore 2.0 software (http://rexhoover.com/index.php?id=cometscore). One-way ANOVA test was used for analysis of the statistical significance and
We studied the effect of a low dose of silibinin on modulating the IR effects in PCa cells. We treated radioresistant DU145 cells with silibinin (25 µM) and/or IR (5 Gy) for the colony formation assay. We observed the synergistic effect (CDI value = 0.83) of combined treatment with silibinin and IR on the reduction in the clonogenic potential of DU145 cells (Fig. 1A and 1B). Treatment with silibin alone induced moderate (24.79%) inhibition of colony formation ability; however, IR alone treatment strongly inhibited (74.4%,
Further, the effect of silibinin and IR on cell proliferation was assessed by the trypan blue assay after the 48-hour treatment. Silibinin and IR reduced the total number of cells by 5.4% and 26.3% (
In an attempt to gain mechanistic insights, we assessed the effect of silibinin on the expression of Rad51, a key protein involved in the HR pathway in DU145 cells. Silibinin (25-100 µM) showed a decreased expression of Rad51 protein in concentration- as well as time-dependent manner after the 12- and 24-hour of treatments (Fig. 1E and 1F). Collectively, these results suggested that a non-toxic low dose of silibinin enhanced the inhibitory effects of IR on cell proliferation and clonogenicity, and also downregulated the expression of Rad51 in radioresistant DU145 cells.
We assessed the effect of silibinin on the biological effects of IR in EGFR-knockdown DU145 cells. We used pLKO.1 and EGFR-knockdown DU145 cells to assess the effect of silibinin after IR exposure on cell proliferation and clonogenic potential. EGFR-knockdown decreased the total cell number in all the treatments. Further, EGFR-knockdown decreased the survival rate by 29% (
Further, silibinin alone was found to decrease the colony formation irrespective of EGFR status of the cells. However, this effect was strongly reduced in EGFR-knockdown cells. Similar effects were observed in cells with IR alone or silibinin + IR treatments with respect to EGFR status of the cells. Silibinin slightly increased the effect of IR in reduction of colony formation ability in EGFR-knockdown cells (Fig. 2C). EGFR-knockdown alone reduced the total number of colonies by 12.3% when compared to vector control. Silibinin augmented the IR-induced effect and decreased the total number of colonies moderately by 16% in EGFR-knockdown cells when compared to IR alone treatment (Fig. 2D). Further, the decrease in cell growth and proliferation in the combined treatment of silibinin and IR in EGFR-knockdown cells was supported by the strong reduction in PCNA protein expression level (Fig. 2E). Collectively, a low dose of silibinin enhanced the IR-induced inhibition of cell growth, cytotoxicity and reduction in clonogenic potential. Moreover, both silibinin and IR were able to exert anticancer effects in EGFR-deficient PCa cells.
Radiotherapy halts the cell cycle progression in tumor cells, and hence IR-induced cell cycle arrest is critical to therapeutic responses. Our study has shown that EGFR-knockdown in DU145 cells increased the G2/M phase cell population moderately. The IR treatment increased the G2/M phase cell population from 23.6% in vector control to 29.2% (
Next, we evaluated the effect of combination of a low dose of silibinin and IR on DNA damage in EGFR-knockdown DU145 cells using an alkaline comet assay. We observed that EGFR-knockdown sensitized the radioresistant PCa cells for DNA damage upon treatment with a low dose of silibinin (25 µM) as well as IR (5 Gy) (Fig. 4A). IR-induced comet tail length in EGFR-knockdown cells increased significantly (
Further, we assessed the role of key molecular proteins involved in DNA DSBs repair pathways, HR and NHEJ. We observed that the silibinin treatment alone suppressed the expression of DNA-PK, a critical protein involved in the NHEJ pathway, in parent DU145 cells when compared to control (Fig. 5A). Silibinin also decreased the protein levels of DNA-PK and Rad51 in EGFR-knockdown cells; however, the levels of Ku70 and Ku80 proteins remained unchanged (Fig. 5A). The combination of silibinin and IR caused a strong decrease in the levels of DNA-PK and Rad51 in EGFR-knockdown cells. Further, silibinin enhanced the IR-induced phosphorylation of p53 protein at serine 15 in EGFR-knockdown DU145 cells, indicating the augmented DNA damage and cell cycle arrest in EGFR-knockdown cells (Fig. 5A). The observed decrease in the total p53 protein level in IR and combination treatments may be indicative of its degradation after the phosphorylation. Hence, in the absence of EGFR signaling, the lower dose of silibinin can enhance the IR-induced DNA damage by blocking HR and NHEJ repair pathways.
Next, we assessed the effect of silibinin on the expression of IR-induced pro-survival signaling molecules, Akt, ERK1/2, and STAT3, involved in initiating the survival response in tumor cells in response to radiotherapy which contributes to the development of radioresistance. Treatment with a low dose of silibinin did not show any considerable effect on the activation of Akt, ERK1/2, and STAT3 in parent or EGFR-knockdown DU145 cells (Fig. 5B). Notably, EGFR-knockdown itself decreased the expression of p-ERK1/2 and p-Akt proteins as compared to vector control. The combined treatment of silibinin and IR in EGFR-knockdown cells strongly decreased the levels of p-STAT3 and p-ERK1/2 without any considerable change in their total protein levels (Fig. 5B). These results suggested that a low dose of silibinin potentially attenuated the survival signaling response in cancer cells exposed to radiation which otherwise would have caused a cytoprotective effect in response to radiotherapy.
The central findings in the present study are that silibinin (a) enhanced the anticancer efficacy of IR in EGFR-knockdown DU145 radioresistant PCa cells, (b) targeted the HR pathway by suppressing Rad51 expression, (c) enhanced IR-induced G2/M arrest in EGFR-knockdown cells, (d) augmented IR-induced DNA damage by (e) attenuating the expression of IR-induced critical DSBs DNA repair proteins including Rad51 and DNA-PK, and (f) in combination with IR inhibited pro-survival signaling molecules including ERK1/2, Akt and STAT3 in EGFR signaling deficient radioresistant PCa cells.
This study deciphered the molecular alterations involved in silibinin-mediated radiosensitizing effects in EGFR-deficient radioresistant PCa cells which was mediated through the suppression of DNA repair pathways. As the role EGFR in promoting of IR-induced DNA repair pathways is known, we studied the potential of silibinin in sensitizing the PCa cells deficient in EGFR signaling. In this study, metastatic prostate carcinoma cells, DU145, were used to delineate the mechanisms associated with phytochemical-induced sensitizing effects on PCa owing to high radioresistant trait as compared to PC-3 and LNCaP cells as reported earlier [15,23].
Our previous study has shown that silibinin, at a low dose, potentially radiosensitized the PCa cells to the ionizing radiation . In agreement with these findings, we observed the augmentation in reduction of clonogenicity in DU145 cells in combination of a low dose of silibinin and IR. Concurrently, silibinin enhanced the IR-induced cytotoxicity in radioresistant PCa cells, and decreased the cell proliferation. Further, we investigated the biological event as how silibinin executes its radiosensitizing effects in PCa cells. IR-mediated DNA damage induction is an integral mechanism for its anticancer effects, as effectiveness of radiotherapy mainly lies on its ability to cause lethal DNA DSBs damage in cancer cells; however, IR-induced DDR machinery activation is a major limitation attributed to the efficient DNA damage repair system possessed by cancer cells . Many studies have revealed the role of HR DNA repair pathway in the development of radioresistance in tumor cells because fast proliferating cancer cells are more dependent on this pathway for survival [25,26].
Further, studies have also defined the role of Rad51, a key homologous recombination protein, in therapeutic resistance associated with poor patient survival to chemo- and radiotherapy and advocated that targeting Rad51 could potentially radiosensitized tumor cells [27-29]. In the present study, we observed that lower doses of silibinin reduced the expression of Rad51 protein in radioresistant DU145 cells. Hence, our study suggested the novel role of silibinin in blocking the Rad51-mediated HR pathway in radioresistant PCa cells.
Our recent study has shown that EGFR regulates intrinsic radioresistance in PCa cells via regulation of DNA repair pathway proteins, such as Rad51 and DNA-PK . Therefore, we delved into the mechanistic aspects of modulation of radiation response by silibinin in the context of EGFR signaling. We found that silibinin synergistically inhibited the cell proliferation and clonogenic potential in EGFR-knockdown DU145 cells along with further augmentation of IR-induced cytotoxicity in cells. This was supported by the decreased expression of PCNA in silibinin-treated EGFR-knockdown PCa cells. It is well established fact that the outcome of the radiotherapy or radiosensitivity of the cancer cells may vary in the different phases of the cell cycle . Silibinin was found to enhance the IR-induced G2/M arrest in EGFR-knockdown DU145 cells, which is clinically relevant as blocking cell cycle in G2/M phase will enhance IR-induced cytotoxic effects in a subsequent cycle of radiation owing to most radiosensitive phase of the cell cycle .
IR-induced EGFR signaling mediates the DNA DSBs damage repair pathways, HR and NHEJ, as well as pro-survival pathways in tumor cells which confers cytoprotective advantages and thus reduces the efficacy of clinical radiotherapy [31-33]. Consistent with these reports, our findings championed the role of receptor tyrosine kinase by regulating DNA repair pathways as blocking EGFR signaling increased the IR-induced DNA damage in radioresistant PCa cells. Nevertheless, silibinin further increased the IR-induced DNA damage in PCa cells in the absence of EGFR signaling. This observation suggested that silibinin can also alter pathways other than EGFR in sensitizing the PCa cells for IR-induced DNA damage. It may be noted that silibinin suppressed the IR-induced expression of critical DSBs repair proteins, Rad51 and DNA-PK which may be regulated by many upstream molecules including EGFR. This observation suggested that silibinin also has the capability of blocking HR and NHEJ pathways independently of EGFR in response to IR-induced DNA damage in radioresistant PCa cells. Concurrently, we did not observe any change in the expression of Ku70 and Ku80 proteins in our experimental conditions. Our study has indicated that the regulation of Ku proteins is independent of EGFR signaling in DU145 cells, as also reported in our previous finding . Of note, the radiosensitizing effect of silibinin in EGFR-knockdown DU145 cells is mediated by targeting DNA-PK and Rad51 proteins, but not the Ku proteins.
The activation of p53 protein in response to IR-induced DNA damage increases its transcriptional activity for various genes involved in DNA repair, cell cycle arrest and apoptosis [34-36]. However, p53 mutations in the DNA binding domain in DU145 cells render p53 transcriptionally inactive  Thus, silibinin as well as IR caused an increase in the phosphorylation of p53 (ser15) irrespective of EGFR status of the cells which was further increased in the combination treatment.
Notably, there was a corresponding decrease in the total p53 protein level which is indicative of its reduced expression or enhanced degradation. These observations suggested that p53 activation is independent of EGFR status of PCa cells and corresponded to the level of DNA damage as well as cell death.
Notably, radiotherapy also activates the pro-survival signaling pathways in tumor cells which play an important role in emergence of radioresistance. The activation of ERK1/2, Akt and STAT3 signaling pathways in response to radiation therapy has been reporeted [38-41]. In the present study, EGFR-knockdown decreased the activation of Akt and ERK1/2 but not the STAT3, suggesting an EGFR-independent regulation of STAT3 in PCa cells. Further, silibinin combination with IR suppressed the activation of STAT3 proteins only in EGFR-deficient cells suggesting that in the absence of EGFR, silibinin may sensitize the PCa cells exposed to IR through down-regulation of STAT3. The silibinin also sensitized the PCa cells exposed to IR through inhibition of ERK1/2 and Akt signaling in EGFR-deficient cells suggesting a further downregulation of pro-survival signaling. Moreover, activated Akt and STAT3 are known to upregulate Rad51 thus suggesting their role in DNA repair which was inhibited by silibinin and IR combination (Fig. 6).
In conclusion, we report the novel molecular alterations associated with a low dose silibinin-mediated radiosensitizing effects in PCa cells by targeting HR and NHEJ pathways. Silibinin mitigated the IR-induced DNA repair through Rad51 and DNA-PK and sensitized the cells to death which was more effective in EGFR-deficient cells. This radiosensitizing effect of silibinin was further supported by the downregulation of pro-survival signaling pathways, ERK1/2, Akt and STAT3, in PCa cells. Together, these findings suggested that the combination approach of targeting EGFR signaling along with the application of silibinin in radioresistant PCa cells could be a better strategy to enhance the radiotherapeutic index in clinical settings.
We are thankful for the technical assistance for flow cytometry, fluorescence microscopy and Gamma irradiation chamber from the Central Instrumentation Facility (CIF) at School of Life Sciences, Jawaharlal Nehru University, India.
Mohit Rajput, Deepali Mishra and Kunal Kumar were supported by Fellowships from University Grant Commission (UGC), India. The work was supported in part by DST, UGC-DRS and RNW, UPE-2 and DST-PURSE, DST-DPRP, India are gratefully acknowledged.
No potential conflicts of interest were disclosed.
Hachung Yoon, Aesun Shin and Keun-Young YooJournal of Korean Association of Cancer prevention 2002; 7(1): 34-41