Ophiopogonin D' Inhibits Prostate Cancer Cell Growth In Vitro And In Vivo, Through Activating The RIP1/MLKL Pathway

Background and Objectives:Human cancers pose serious public health and socio-economic problems worldwide,with increasing cancer incidence and mortality.It is estimated that in China,there are about 4,292,000 new cancer cases and 2,814,000 cancer deaths in 2015.Prostate cancer is the most frequently diagnosed cancer among men in developed countries.Although Asia and China belong to low incidence areas,the incidence of prostate cancer increased 4.7% year-on-year from 2005~2011 in China,and mortality increased 5.5% year-on-year from 2000 to 2011.Aging is a recognized risk factor for prostate cancer,with more than 50% incidence and more than 90% mortality rate of prostate cancer occurring in men over 65 years of age.With a continued increase in life expectancy of Chinese men and increase in aging population,it is expected that both incidence and mortality of prostate cancer will increase,becoming one of the important diseases that threat men's health.Most prostate cancer cases are androgen-dependent,and hormone therapy(mainly through androgen deprivation)represents one of the mainstay treatment options.Androgen-deprivation therapy(ADT)can prevent prostate cancer progression though inhibiting androgen,but is often associated with a variety of complications,such as hot flushes,loss of libido,muscle loss,fatigue,cognitive dysfunction,and depression.Long-term adverse effects of ADT include osteoporosis,anemia,and increased risk of metabolic syndrome,cardiovascular disease,and death.What's more,not all patients respond to ADT,and those initially responding to ADT become resistant or refractory,with the median response duration being only 18 months and most prostate cancer patients developing into castrated-resistant prostate cancer(CRPC).CRPC patients are usually with high mortality,and chemotherapy is still the main treatment of CRPC,but with poor clinical outcome.Therefore,it is particularly important to develop novel therapy for CRPC.Natural compounds represent rich sources for discovery and development of novel anticancer agents;they are biologically active small molecules and often produced by plants,animals,microorganisms,and marine organisms.In fact,more than 60% of the anticancer drugs are derived from natural products.Natural compounds often afford lead compounds that can be developed to clinical candidate anticancer drugs;so that natural compounds may be composed into anti-tumor drug library.Among natural anticancer agents,saponins compounds have been shown to be active against prostate cancer,through inhibiting proliferation,inducing apoptosis and cell cycle arrest.Previous studies from our laboratory have demonstrated that Platycodin D,derived from Platycodin grandiflorum,inhibits cell proliferation,induces apoptosis,and arrests cell cycle in vitro and inhibits tumor growth in vivo.This thesis research was designed to investigate the anticancer activity of Ophiopogonin D'(OPD'),an analog of Platycodin D,and explore the underlying mechanism of action.Since its conceptual inception in 2005,necroptosis has attracted cancer researchers to focus on its role in tumor cell death and potential as a drug target.Although necroptosis is characterized by both necrotic morphological features(e.g.,cell swelling and disappearance of cell membrane integrity)and gene regulation patterns like apoptosis,it is not affected by caspase inhibitors but can be inhibited by necrostatin 1(Nec-1),which is necroptosis specific inhibitors,targeted receptor-interacting protein 1(RIP1).It is reported that the mixed lineage kinase domain-like protein(MLKL)is a core molecule in the process of necroptosis.When the cells undergo necroptosis,MLKL phosphorylation causes oligomerization of the monomer MLKL,and the oligomerized MLKL migrates from the cytoplasm to the cell membrane to induce necroptosis.Necroptosis has been reported to play an important role in the induction of cell death in various malignancies such as colon cancer,lymphoma,and oral cancer.Tea polyphenols,extracted from green tea,have been found to induce necroptosis in prostate cancer PC3 cells.Ophiopogonin D'(OPD'),a saponin compound extracted from ophiopogon japonicus,has not been studied for its effects on prostate cancer.In the present study,we aimed at demonstrating the anticancer effects of OPD' on PC3 cells in vitro and in vivo and exploring its mechanism of action.In our in vivo studies with nude mouse xenograft model,the safety profile of OPD' was also evaluated.Methods:The MTT assay was performed to determine the effects of the OPD' on the viability of prostate cancer PC3 cells.The effects of OPD' on apoptosis/necrosis were performed with Annexin V-FITC and PI double staining in PC3 cells by flow cytometry.In order to clarity whether the OPD'-induced cell death is caspase-dependent or not,we used inhibitors of caspase--Z-VAD-FMK and AC-DEVD-CHO in the in vitro assays.Trypan blue and lactate dehydrogenase(LDH)released assays were used to evaluate the effects of OPD' on cell membrane integrity,which was further verified by the acridine orange/ethidium bromide(AO/EB)staining experiment.Meanwhile,crystal violet staining and transmission electron microscopy were performed to investigate the effects of OPD' on cell morphological changes.Moreover,necrostatin 1(Nec-1),an inhibitor of necroptosis,was used to verify the OPD'-induced necroptosis.The protein levels of cleaved-RIP1,RIP1,p-MLKL,and MLKL were detected by Western blotting analyses.The maximum tolerated dose(MTD)was determined for selection of the dose in the nude mice with transplanted tumors as well as toxicity testing.Finally,PC3 xenograft tumors were established in BALB/c nude mice and used for testing of the anti-tumor effects of OPD' in vivo.Results:OPD' showed significant anticancer effects against PC3 cells in vitro,with the IC50 value being 6.25 ± 0.31 ?M.Annexin V-FITC and PI double staining showed that OPD' mainly induced PC3 cells late apoptosis/necrosis;the treatment with 5 ?M of OPD' for 6 hr induced late apoptosis/necrosis in a time-dependent manner(r = 0.728,P < 0.001).Based on the results from the assays with caspase inhibitors,the OPD'-induced cell death was necroptosis-dependent,but not caspase-dependent.Trypan blue and LDH released assays showed that PC3 cells were stained positive and release of LDH was increased followig 5 ?M OPD' treatment.AO/EB staining also confirmed that 5 ?M OPD' induced damages to cell membrane integrity.In addition,morphological observation demonstrated that OPD' induced cell swelling,nucleolus dissolution,morphological necrosis-like changes,which were consistent with morphological changes of necroptosis.The treatment with 5 ?M of OPD' for 6 hr significantly down-regulated cleaved-RIP1 and up-regulated RIP1,resulting in a decrease in cleaved-he RIP1/RIP1 ratio.These results indicated that OPD' induced prostate cancer cell necroptosis through regulating RIP1.Western blotting analyses showed that OPD' significantly up-regulated p-MLKL(220 KD),but not MLKL,resulting in an increase in the p-MLKL/MLKL ratio,indicating that OPD' induced prostate cancer cell necroptosis through regulating p-MLKL.The MTD assay showed that 20 mg/kg OPD' was tolerable in normal Kunming mice,and the MTD for nude mice was 5 mg/kg.Compared to the control group,5 mg/kg OPD' group led to 79.79% inhibition of the tumor growth of PC3 xenografts,with a complete regression being observed in three nude mice.What's more,5 mg/kg OPD' treatment did not induce significant decrease in body weight in the nude mice,indicating that OPD' had no obvious side effects in BALB/c nude mice.Conclusions:Our results demonstrated that OPD' effectively inhibited the survival of androgen-independent PC3 cells in vitro,which was mediated by necroptosis through regulating the RIP1/MLKL pathway.OPD' significantly inhibited the growth of PC3 xenografts with tumor regression being found in some of the treated tumor-bearing nude mice.Additionally,OPD' did not produce significant side effects at the effective dose in vivo.Taken together,OPD' showed strong in vitro and in vivo anti-prostate cancer activity with reasonable safety profile,suggestion that it could be developed as a potential clinical candidate for prostate cancer therapy.