Appotosis Inducing Effects Of Cinobufacini And Its Active Compounds Bufalin And Cinobufagin

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant neoplasms worldwide and its incidence has been increasing over the past few decades in some areas such as Europe, the US, and East Asia. Although surgical resection, liver transplantation, and radiofrequency ablation are common options for HCC, patients with advanced HCC are not candidates for these therapies. In addition, because of the inherently chemotherapyresistant nature of HCC, systemic cytotoxic chemotherapy agents are minimally effective at improving patient survival. Thus, a major goal is to find new chemotherapeutic agents and more effective therapies for the treatment of HCC.Recently, several traditional Chinese medicines with anti-tumor properties have attracted considerable interest as candidates for the development of novel cancer therapeutics. Cinobufacini is a water-soluble extract from the skins and parotid venom glands of the toad Bufo bufo gargarizans Cantor and has been used in China as an effective traditional Chinese medicine to treat conditions like swelling, pain, and heart failure for thousands of years. Currently, cinobufacini, as a form of traditional Chinese medicine has been approved by the Chinese State Food and Drug Administration (SFDA) (ISO9002), and widely used to treat patients with liver, lung, colon and pancreatic cancers at oncology clinics in China. Bufadienolides, a class of cardioactive C-24 steroids with a characteristic a-pyrone ring at C-17, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. Since bufadienolides are the maker biologically active compounds of cinobufacini, the antitumor activity of cinobufacinimaybe due to this kind of components. Recently, many experimental and clinical studies on cinobufacini and some of its active compounds have been carried out around theworld to investigate their antitumor activity and mechanisms. Numerous in vitro and in vivo datasets have suggested that cinobufacini and its active compounds bufalin and cinobufagin exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data showed that cinobufacini used alone or in combination with other chemotherapeutic agents (e.g. gemcitabine and oxaliplatin) had significant anticancer activity against human cancers, such as hepatocellular carcinoma, nonsmall-cell lung cancer, pancreatic cancer, and gallbladder carcinoma. These indicated that cinobufacini and its active compounds bufalin and cinobufagin may have effective anticancer activity with low toxicity and few side effects. However, the pharmacologic constituents of cinobufacini still need further study and the precise mechanisms of cinobufacini and its active compounds bufalin and cinobufagin in HCC cells are still not clear. Thus, it is important to screen and identify the active compounds of cinobufacini and investigate the mechanisms of cinobufacini and its active compounds bufalin and cinobufagin in HCC to provide additional evidence for HCC treatment.Apoptosis, or programmed cell death, is a normal physiological process that plays an important role in many normal functions ranging from embryonic development to adult tissue homeostasis. Cancer is characterized by uncontrolled cell proliferation and deregulation of apoptosis, which represents a major protective mechanism against cancer. Therefore, activation of the apoptotic pathway in tumor cells is considered to be a protective mechanism against the development and progression of cancer. There are two main signaling pathways that are involved in apoptosis, the extrinsic and the intrinsic pathway, which both result in the activation of caspases. The extrinsic pathway, also called death receptor-mediated apoptosis pathway, is activated by ligand-bound death receptors of the tumor-necrosis factor receptor (TNFR) superfamily (e.g. Fas, TNF-R1 and Apo-2). Stimulation of Fas has been shown to be highly efficient in the killing of tumor cells. At the cell surface, Fas receptor binding to its ligand leads to the formation of the death-inducing signal complex, which contains the adaptor protein Fas-associated death domain protein (FADD) and caspase-8 and -10. FADD leads to the auto-cleavage and activation of caspase-8 and-10. A large amount of active caspase-8 and -10 in turn activates downstream caspases such as caspase-3, causing apoptosis. A small amount of active caspase-8 and -10 cleaves the BH-3-only protein Bid into tBid, which subsequently triggers the release of cytochrome c and activation of the mitochondriamediated apoptosis pathways. The intrinsic pathway, also called the mitochondria-mediated apoptosis pathway, is a signal transduction pathway involving the mitochondria and the Bcl-2 family. The Bcl-2 family proteins Bax and Bcl-2 play important roles in initiating the mitochondrial death cascade. Pro-apoptotic protein Bax translocates to the mitochondria and integrates into the outer mitochondrial membrane, where it promotes the disruption of mitochondrial membrane potential and the release of cytochrome c into the cytosol. In contrast, anti-apoptotic protein Bcl-2 prevents this process by preserving mitochondrial integrity. Thus, the ratio of Bax to Bcl-2 is crucial to the sustenance of drug-induced apoptosis in the mitochondria-mediated apoptotic pathway. Following the disruption of Dwm and the release of cytochrome c, a caspase cascade including caspase-9 and caspase-3 is activated. Activated caspase-3 subsequently cleaves intracellular protein PARP into cleaved PARP, which serves as an important marker of apoptosis. Moreover, BH-3-only protein Bid is considered to be a molecular linker bridging the death receptor pathway and mitochondria pathway. Once Bid is cleaved by active caspase-8 and caspase-10 as described above, tBid translocates to mitochondria and induces mitochondrial damage and release of cytochrome c. Although cinobufacini and its active compounds bufalin and cinobufagin have exhibited great apoptosis-inducing effect on some cancers, the precise mechanisms of these reagents in HCC cells are still not clear. Thus, in the present study, the active compounds of cinobufacini were screened and identified, and the possible pathways and related molecular mechanisms by which cinobufacini and its active compounds bufalin and cinobufagin induce apoptosis in the HCC cells are investigated. Each part is described as follows:Part I. Screening and identifying anti-hepatocarcinoma active compounds from cinobufaciniAim of the study:The present study sought to screen and identify active compounds from cinobufacini and investigate their anti-hepatocarcinoma effect on HepG2 cells.Materials and Methods:The effects of different fractions on the proliferation of HepG2 cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. Moreover, the cytotoxic activity of these two active compounds on HepG2 cells was further detected by MTT assay.Results:Through bioassay-guided isolation, two effective anti-cancer components from cinobufacini were purified. TLC and HPLC results indicated that they were two highly homogeneous compounds.1H NMR,13C NMR, DEPT, and EI-MS data of these two compounds were obtained. After analyzing and comparing these data online (SciFinder Scholar) with reported compounds and references, the structure of these two compounds were identified as cinobufagin and resibufogenin. Furthermore, MTT assay indicated that the cytotoxic activity of cinobufagin was greater than that of resibufogenin in HepG2 cells.Conclusion:Two compounds, resibufogenin and cinobufagin, were isolated from cinobufacini. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells.Partâ…¡. Apoptosis-inducing effect of cinobufacini on HCC cell lines HepG2 and BEL-7402Aim of the study:The present study sought to investigate the possible signaling pathway implicated in cinobufacini duced apoptosis in the hepatocellular carcinoma cell lines HepG2 and Bel-7402.Materials and Methods:The effects of cinobufacini on cell proliferation of HepG2 and Bel-7402 cells were evaluated by MTT assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential and caspase-3 and caspase-9 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins (Bax, Bcl-2, Fas, Bid, caspase-3, caspase-9, poly(ADP-ribose)polymerase (PARP), and cleaved PARP) and release of cytochrome c were assessed by Western Blot analysis.Results:Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and timedependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP).Conclusion:The present study indicated that cinobufacini can induce apoptosis of HepG2 and Bel-7402 cells through mitochondria-mediated apoptotis pathways. Partâ…¢. Apoptosis-inducing effect of bufalin and cinobufagin on HCC cells HepG2Aim of the study:The present study sought to investigate the possible signaling pathway implicated in bufalin and cinobufagin duced apoptosis in the hepatocellular carcinoma cells HepG2.Materials and Methods:The effects of bufalin and cinobufagin on cell proliferation of HepG2 cells were evaluated by MTT assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential and caspase-3, caspase-9, caspase-8, and caspase-10 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins (Bax, Bcl-2, Fas, Bid, caspase-3, caspase-9, caspase-8, caspase-10, poly(ADP-ribose)polymerase (PARP), and cleaved PARP) and release of cytochrome c were assessed by Western Blot analysis. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVDFMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHDFMK and caspase-10 inhibitor Z-AEVD-FMK.Results:Bufalin and cinobufagin significantly inhibited cell proliferation of HepG2 cells in a dose- and timedependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after bufalin and cinobufagin treatment. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3,-8,-9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVDFMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHDFMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor.Conclusion:The present study demonstrated that bufalin and cinobufagin induced apoptosis of HepG2 cells via both Fas- and mitochondria-mediated apoptotic pathways (Fig.10). Caspase-3,-8,-9 and -10 all took part in regulation of these two pathways and a caspase-10-dependent Fas-mediated pathway might play a critical role. These findings should provide important clues for further evaluating the potential potency of bufalin and cinobufagin for use in HCC therapy.