Isolation, Identification and Chemical Modification of Bufadienolides from Bufo melanostictus Schneider and Their Cytotoxic Activities against Prostate Cancer Cells.

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3-13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Arenobufagin enhances T-cell anti-tumor immunity in colorectal cancer by modulating HSP90ß accessibility.

BACKGROUND: Colorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin (Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. PURPOSE: This study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90ß-STAT3-PD-L1 axis activity. METHODS: We investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits CRC. Utilizing target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90ß (HSP90ß) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. RESULTS: We observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90ß's accessibility significantly decreased upon Are binding. We demonstrated a decrease in the activity of the HSP90ß-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. CONCLUSION: Contrary to previous research focusing on the direct cytotoxicity of Are towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Are and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Are.

Arenobufagin modulation of PCSK9-mediated cholesterol metabolism induces tumor-associated macrophages polarisation to inhibit hepatocellular carcinoma progression.

BACKGROUND: The tumor microenvironment (TME) of hepatocellular carcinoma is heterogeneous enough to be prone to drug resistance and multidrug resistance during treatment, and reprogramming of cholesterol metabolism in TME mediates tumor-associated macrophages (TAMs) polarization, which has an impact on the regulation of malignant tumor progression. Arenobufagin (ARBU) was extracted and isolated from toad venom (purity ≥98 %), which is the main active ingredient of the traditional Chinese medicine Chan'su with good anti-tumor effects. PURPOSE: To investigate the regulatory effect of ARBU on lipid metabolism in tumor microenvironment, interfere with macrophage polarization, and determine its mechanism of action on liver cancer progression. METHODS: In this study, the inhibitory effect of ARBU on the proliferation of Hepa1-6 in C57 mice and the safety of administration were evaluated by establishing a transplanted tumor model of Hepa1-6 hepatocellular carcinoma mice and using 5-FU as a positive control drug. In addition, we constructed a co-culture system of Hepa1-6 cells and primary mouse macrophages to study the effects of ARBU on the polarization phenotypic transformation of macrophages and the proliferation and migration of hepatoma cells. The influence of ARBU on the metabolism of lipids in the hepatocellular carcinoma mouse model was investigated by combining it with lipidomics technology. The influence of ARBU on the PCSK9/LDL-R signaling pathway and macrophage polarization, which regulate cholesterol metabolism, was tested by using qRT-PCR, gene editing, IF, and WB. CONCLUSION: ARBU significantly inhibited the proliferation of Hepa1-6 in vivo and in vitro, regulated cholesterol metabolism, and promoted the M1-type polarization of macrophages in the tumor microenvironment. ARBU inhibits cholesterol synthesis in the TME through the PCSK9/LDL-R signaling pathway, thereby blocking macrophage M2 polarization, promoting apoptosis of the tumor cells, and inhibiting their proliferation and migration.

Arenobufagin, isolated from Bufo viridis toad venom, inhibits A549 cells proliferation by inducing apoptosis and G2/M cell cycle arrest.

Lung cancer is a significant contributor to cancer morbidity and mortality globally. Arenobufagin, a compound derived from Bufo viridis toad venom, has demonstrated the ability to inhibit cell growth in various cancer cell lines. However, our understanding of the role and mechanism of arenobufagin in lung cancer remains incomplete, necessitating further researches to fully elucidate its action mechanism. In this study, we further explored the impact of arenobufagin on A549 cells. The results revealed that it exerted a potent cytotoxic effect on A549 cells by inhibiting cell colony formation, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels, and arresting A549 cells in G2/M phase. Collectively, our findings suggested that arenobufagin may have potential as a future therapeutic for lung cancer treatment.

Chemical Composition, Pharmacological Effects and Clinical Applications of Cinobufacini.

Chinese medicine cinobufacini is an extract from the dried skin of Bufo bufo gargarizans Cantor, with active ingredients of bufadienolides and indole alkaloids. With further research and clinical applications, it is found that cinobufacini alone or in combination with other therapeutic methods can play an anti-tumor role by controlling proliferation of tumor cells, promoting apoptosis, inhibiting formation of tumor neovascularization, reversing multidrug resistance, and regulating immune response; it also has the functions of relieving cancer pain and regulating immune function. In this paper, the chemical composition, pharmacological effects, clinical applications, and adverse reactions of cinobufacini are summarized. However, the extraction of monomer components of cinobufacini, the relationship between different mechanisms, and the causes of adverse reactions need to be further studied. Also, high-quality clinical studies should be conducted.

Pharmacological Effects of Cinobufagin.

Cinobufagin (CBF) is a bufadienolide, which is a major active ingredient of toad venom. In recent years, CBF has attracted increasing attention due to its highly potent and multiple pharmacological activities. To better understand the status of research on CBF, we collated recent studies on CBF to provide a valuable reference for clinical researchers and practitioners. According to reports, CBF exhibits extensive pharmacological properties, including antitumor, analgesic, cardioprotection, immunomodulatory, antifibrotic, antiviral, and antiprotozoal effects. Studies on the pharmacological activity of CBF have mainly focused on its anticancer activity. It has been demonstrated that CBF has a therapeutic effect on liver cancer, osteosarcoma, melanoma, colorectal cancer, acute promyelocytic leukemia, nasopharyngeal carcinoma, multiple myeloma, gastric cancer, and breast cancer. However, the direct molecular targets of CBF are currently unknown. In addition, there are few reports on toxicological and pharmacokinetic of CBF. Subsequent studies focusing on these aspects will help promote the development and application of CBF in clinical practice.

The Effects of Cinobufagin on Hepatocellular Carcinoma Cells Enhanced by MRT68921, an Autophagy Inhibitor.

Cinobufagin, a cardiotonic steroid derived from toad venom extracts, exhibits significant anticancer properties by inhibiting Na[Formula: see text]/K[Formula: see text]-ATPase in cancer cells. It is frequently used in clinical settings to treat advanced-stage cancer patients, improving their quality of life and survival time. However, its long-term use can result in multidrug resistance to other chemotherapy drugs, and the exact mechanism underlying this effect remains unknown. Therefore, this study explores the molecular mechanism underlying the anticancer effects of cinobufagin in hepatocellular carcinomas (HCCs), specifically in HepG2 and Huh-7 cells. As determined using transcriptome analysis, cinobufagin-triggered protective autophagy suppressed cell apoptosis in liver cancer HepG2 and Huh-7 cells by inhibiting the phosphoinositide-3-Kinase (PI3K)-AKT serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR) pathway. Cinobufagin-inhibited cell proliferation, induced apoptosis, and generated cell autophagy by upregulating the expression of MAP1 light chain 3 protein II, Beclin1, and autophagy-related protein 12-5. In addition, the autophagy inhibitor MRT68921 improved the antiproliferative and proapoptotic effects of cinobufagin in the studied cell lines. Overall, this study suggests that combining cinobufagin with an autophagy inhibitor can effectively treat HCC, providing a potential strategy for cancer therapy.

Toad venom bufadienolides and bufotoxins: An updated review.

Bufadienolides, naturally found in toad venoms having steroid-like structures, reveal antiproliferative effects at low doses. However, their application as anticancer drugs is strongly prevented by their Na+ /K+ -ATPase binding activities. Although several kinds of research were dedicated to moderating their Na+ /K+ -ATPase binding activity, still deeper fundamental knowledge is required to bring these findings into medical practice. In this work, we reviewed data related to anticancer activity of bufadienolides such as bufalin, arenobufagin, bufotalin, gamabufotalin, cinobufotalin, and cinobufagin and their derivatives. Bufotoxins, derivatives of bufadienolides containing polar molecules mainly belonging to argininyl residues, are reviewed as well. The established structures of bufotoxins have been compiled into a one-page figure to review their structures. We also highlighted advances in the structure-modification of the structure of compounds in this class. Drug delivery approaches to target these compounds to tumor cells were discussed in one section. The issues related to extraction, identification, and quantification are separated into another section.

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest.

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.

Uncovering the Mechanisms of Active Components from Toad Venom against Hepatocellular Carcinoma Using Untargeted Metabolomics.

Toad venom, a dried product of secretion from Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, has had the therapeutic effects of hepatocellular carcinoma confirmed. Bufalin and cinobufagin were considered as the two most representative antitumor active components in toad venom. However, the underlying mechanisms of this antitumor effect have not been fully implemented, especially the changes in endogenous small molecules after treatment. Therefore, this study was designed to explore the intrinsic mechanism on hepatocellular carcinoma after the cotreatment of bufalin and cinobufagin based on untargeted tumor metabolomics. Ultraperformance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was performed to identify the absorbed components of toad venom in rat plasma. In vitro experiments were determined to evaluate the therapeutic effects of bufalin and cinobufagin and screen the optimal ratio between them. An in vivo HepG2 tumor-bearing nude mice model was established, and a series of pharmacodynamic indicators were determined, including the body weight of mice, tumor volume, tumor weight, and histopathological examination of tumor. Further, the entire metabolic alterations in tumor after treating with bufalin and cinobufagin were also profiled by UHPLC-MS/MS. Twenty-seven active components from toad venom were absorbed in rat plasma. We found that the cotreatment of bufalin and cinobufagin exerted significant antitumor effects both in vitro and in vivo, which were reflected in inhibiting proliferation and inducing apoptosis of HepG2 cells and thereby causing cell necrosis. After cotherapy of bufalin and cinobufagin for twenty days, compared with the normal group, fifty-six endogenous metabolites were obviously changed on HepG2 tumor-bearing nude mice. Meanwhile, the abundance of α-linolenic acid and phenethylamine after the bufalin and cinobufagin intervention was significantly upregulated, which involved phenylalanine metabolism and α-linolenic acid metabolism. Furthermore, we noticed that amino acid metabolites were also altered in HepG2 tumor after drug intervention, such as norvaline and Leu-Ala. Taken together, the cotreatment of bufalin and cinobufagin has significant antitumor effects on HepG2 tumor-bearing nude mice. Our work demonstrated that the in-depth mechanism of antitumor activity was mainly through the regulation of phenylalanine metabolism and α-Linolenic acid metabolism.

Bufalin for an innovative therapeutic approach against cancer.

Bufalin is an endogenous cardiotonic steroid, first discovered in toad venom but also found in the plasma of healthy humans, with anti-tumour activities in different cancer types. The current review is focused on its mechanisms of action and highlights its very large spectrum of effects both in vitro and in vivo. All leads to the conclusion that bufalin mediates its effects by affecting all the hallmarks of cancer and seems restricted to cancer cells avoiding side effects. Bufalin decreases cancer cell proliferation by acting on the cell cycle and inducing different mechanisms of cell death including apoptosis, necroptosis, autophagy and senescence. Bufalin also moderates metastasis formation by blocking migration and invasion as well as angiogenesis and by inducing a phenotype switch towards differentiation and decreasing cancer cell stemness. Regarding its various mechanisms of action in cancer cells, bufalin blocks overactivated signalling pathways and modifies cell metabolism. Moreover, bufalin gained lately a huge interest in the field of drug resistance by both reversing various drug resistance mechanisms and affecting the immune microenvironment. Together, these data support bufalin as a quite promising new anti-cancer drug candidate.

Cinobufagin induces acute promyelocytic leukaemia cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway.

CONTEXT: Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available. OBJECTIVE: We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells. MATERIALS AND METHODS: We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), ß-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting. RESULTS: CBG inhibited the viability of NB4 and NB4-R1 cells. The IC50 values of NB4 and NB4-R1 cells treated with CBG for 24 h were 45.2 nM and 37.9 nM, respectively. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner and inhibited the ß-catenin signalling pathway. DISCUSSION AND CONCLUSION: CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.

Toad venom: A comprehensive review of chemical constituents, anticancer activities, and mechanisms.

Toad venom, a traditional natural medicine, has been used for hundreds of years in China for treating different diseases. Many studies have been performed to elucidate the cardiotonic and analgesic activities of toad venom. Until the last decade, an increasing number of studies have documented that toad venom is a source of lead compound(s) for the development of potential cancer treatment drugs. Research has shown that toad venom contains 96 types of bufadienolide monomers and 23 types of indole alkaloids, such as bufalin, cinobufagin, arenobufagin, and resibufogenin, which exhibit a wide range of anticancer activities in vitro and, in particular, in vivo for a range of cancers. The main antitumor mechanisms are likely to be apoptosis or/and autophagy induction, cell cycle arrest, cell metastasis suppression, reversal of drug resistance, or growth inhibition of cancer cells. This review summarizes the chemical constituents of toad venom, analyzing their anticancer activities and molecular mechanisms for cancer treatments. We also outline the importance of further studies regarding the material basis and anticancer mechanisms of toad venom.

The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Cinobufacini Inhibits Colon Cancer Invasion and Metastasis via Suppressing Wnt/ß-Catenin Signaling Pathway and EMT.

Cinobufacini is a well-known Chinese medicine extracted from Venenum Bufonis, also called Chan Su. It has been used clinically for various cancers, including colon cancer. However, the function of Cinobufacini on colon cancer invasion and metastasis, and its underlying molecular mechanism, is still not clear. In this study, we investigated the function and mechanism of Cinobufacini on colon cancer invasion and metastasis both in vitro and in vivo studies. Human colon cancer cells were cultured. CCK assay was used to detect the effect of Cinobufacini on colon cancer cells proliferation. The invasion and migration abilities were observed by transwell assays, and the expression of invasion and migration related genes MMP2, MMP9, and epithelial-to-mesenchymal transition (EMT) relate genes were observed by Western blot assays. An orthotopic xenograft model in nude mice was established using colon cancer HCT116 cells, and the function of Cinobufacini on colon cancer invasion and metastasis were observed in vivo. We found Cinobufacini significantly inhibited colon cancer cell proliferation in a dose/time-dependent manner; the invasion and migration abilities of colon cancer were decreased after treated with Cinobufacini. The metastasis and EMT related genes MMP9, MMP2, N-cadherin and Snail were obviously down-regulated, while the expression of E-cadherin was up-regulated after treatment with Cinobufacini. The Wnt/ß-catenin signaling pathway related genes were observed using WB,and results show that the expression of ß-catenin, wnt3a, c-myc, cyclin D1, and MMP7 were all down-regulated after being treated with cinobufacini, while the expression of APC was up-regulated. In vivo studies of the volume and weight of orthotopic xenograft tumors showed significantly shrinkage in the Cinobufacini group compared to the control group. The enterocoelia and liver metastasis tumors were significantly decreased, and the expression of MMP9, MMP2, and ß-catenin were also down-regulated, while E-cadherin was up-regulated in vivo after the treatment with Cinobufacini. Our data proves that Cinobufacini can inhibit colon cancer invasion and metastasis both in vitro and in vivo; the mechanism is related by suppressing the Wnt/ß-catenin signaling pathway and then inhibiting the EMT of CRC.

Cytotoxic and antiproliferative activities of amphibian (anuran) skin extracts on human acute monocytic leukemia cells.

The use of preparations derived from frog skins for curative purposes antedates research history and is perpetuated in current medicine. The skins of anuran's (frogs and toads) are a rich source of compounds with a great importance in the search of antibiotics, analgesics, immunomodulators, enzymatic inhibitors and antitumoral agents applying to human health. Nowadays, cancer is the second most common cause of mortality with more than 8.2 million of deaths worldwide per year. Acute monocytic leukemia is the subtype M5 of acute myeloid leukemia (AML) a cancer type with reduced survival rates in patients. The monocyte to macrophage differentiation plays an essential role increasing the expansion of AML cell lines. Herein we studied the cytotoxic and antiproliferative activities of eleven amphibian species of three families belonging to Argentinean zones, against THP-1 monocytes and THP-1 macrophages acute monocytic leukemia cell lines. The evaluated species showed pronounced deleterious effects on acute monocytic leukemia THP-1 cell lines, reducing cell proliferation and inducing apoptosis, autophagy and in some cases cell aggregation. Being this work of great importance for the study of new natural anti-cancer compounds.

Cinobufacini ameliorates experimental colitis via modulating the composition of gut microbiota.

BACKGROUND: Cinobufacini, the sterilized hot water extraction of dried toad skin, has been widely used in the treatment of inflammation and cancers. Recently we found cinobufacini could ameliorate dextran sulfate sodium (DSS)-induced colitis in mice, but the underlying mechanism was not fully understood. In current study, we explored the effect of cinobufacini on gut microbiota in DSS-induced acute colitic mouse model by pyrosequencing of colonic contents. METHODS: C57BL/6 mice were supplied with normal or 3.0% DSS containing drinking water. DSS-treat mice were gavaged daily either with vehicle (water) or cinobufacini (10.0 or 30.0 mg/kg) for 7 days. The composition of the gut microbiota was assessed by analyzing 16S rRNA gene sequences. RESULTS: Our data indicated that cinobufacini reversed DSS-induced gut dysbiosis and enhanced intestinal barrier integrity. Moreover, changing of some specific microbial groups such as Proteobacteria and Bacteroides was closely correlated with the re-establishment of intestinal equilibrium and the recovery of intestinal function. CONCLUSION: Cinobufacini prevents colitis in mice by modifying the composition and function of gut microbiota. The current study provides additional mechanistic insight in the therapeutic effect of cinobufacini treatment and may pave the way for clinical application of cinobufacini in colitis therapy.

Huachansu Capsule inhibits the proliferation of human gastric cancer cells via Akt/mTOR pathway.

BACKGROUND: For decades, the traditional Chinese medicine preparation, Huachansu Capsule (HCS), has been applied to a variety of solid tumors and leukemias with significant curative effects. More importantly, HCS has few side effects on cardiovascular and gastrointestinal functions in patients. However, the potential mechanism of the anti-tumor activity of HCS has not been fully revealed. The current study investigated the in vivo and in vitro effects of HCS on the proliferation and apoptosis of human gastric cancer (GC) cells and explored the underlying mechanism. MATERIALS AND METHODS: HCS was first diluted to varying concentrations followed by the treatment to MGC-803 and BGC-823 GC cells. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell invasion and migration were assessed using Transwell membrane chambers. Apoptosis and cell cycle arrest in GC cells induced by HCS were detected by flow cytometry. Western blotting assays were used to measure the influence of HCS on apoptosis-related proteins, including B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Cleaved-Caspase-3. Additionally, mammalian target of rapamycin (mTOR) signaling pathway-related proteins such as phosphorylated (p)-Akt, p-mTOR and p-4E-BP1 were detected. Transmission electron microscopy was used to observe the microstructure of apoptotic cells. An animal imaging technique was used to analyze the influence of HCS on the growth of GC cells in vivo and immunohistochemistry assays were performed to investigate the signal transduction pathways influenced by HCS. RESULTS: HCS significantly inhibited the proliferation, invasion and migration of MGC-803 and BGC-823 GC cells. It also induced cell cycle arrest at the G2/M phase and increased the cell apoptotic rate. Additionally, the HCS treatment downregulated the protein levels of Bcl-2, but upregulated the protein expression of Bax and cleaved-caspase 3. Furthermore, HCS downregulated the levels of p-Akt, p-mTOR and p-4E-BP1, suggesting that HCS inhibited tumor growth of GC via suppressing the Akt/mTOR pathway. CONCLUSION: This study indicated that HCS has significant anti-proliferative and apoptotic effects both in vitro and in vivo, and that HCS can inhibit tumor growth of GC via suppressing the Akt/mTOR pathway and induce apoptosis through the intrinsic pathway. Our study provides a scientific basis for the clinical application of HCS.

Bufothionine exerts anti-cancer activities in gastric cancer through Pim3.

AIM: Gastric cancer (GC) is the fourth most common cancer globally. Bufothionine is a major active constituent of Cinobufacini (Huachansu), which is extracted from the skin and parotid venom gland of the toad Bufo bufo gargarizans Cantor. It exhibits anti-cancer activities in vitro. However, whether bufothionine exerts anti-cancer activities against GC is unknown. This study was designed to evaluate the efficacy of bufothionine in vitro and in vivo. MATERIAL AND METHODS: MKN28 and AGS cells were chosen as cell models to study the anti-cancer effect of bufothionine. Cell viability was determined by CCK-8 assay, while the effect of bufothionine on cell membrane integrity was examined by LDH assay. Cell apoptosis was detected by Hoechst/PI staining and Annexin V-FITC/PI staining followed by flow cytometry analysis. The expression levels of proteins involved were examined using western blotting. I-Traq analysis was conducted to identify the differentially expressed genes in AGS cells following bufothionine treatment. The anti-growth effect of bufothionine was validated in vivo using a GC xenograft model. KEY FINDINGS: The results revealed that bufothionine prevented the growth, destroyed cell membrane and promoted apoptotic cell death of GC cells. iTRAQ analysis revealed thatPIM3 might be a molecular target responsible for the anti-cancer effects of bufothionine. It was also found that PIM3 knockdown significantly augmented the anti-growth and pro-apoptotic effects of bufothionine in GC cells. In contrast, ectopic PIM3 expression markedly dampened the anti-neoplastic activities of bufothionine. The expression of PIM3 was also suppressed by bufothionine treatment in xenograft tumor tissue. SIGNIFICANCE: Bufothionine exhibited anti-cancer activities in vitro and in vivo in GC via downregulating PIM3.