Examining the Mechanisms of Huachansu Injection on Liver Cancer through Integrated Bioinformatics Analysis.

OBJECTIVE: The objective of this study is to explore the potential anti-liver cancer mechanism of Huachansu injection through integrated bioinformatics analysis. METHODS: Active ingredients of Huachansu injection (extraction of toad skin) were obtained, and their potential drug targets were predicted via SwissTargetPrediction database. Liver cancer disease targets were identified from the GEO (Gene Expression Omnibus) dataset and four public databases. Then Protein-Protein Interaction (PPI) network of toad skin was constructed. GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were performed subsequently. Finally, molecular docking was performed using Auto Dock Vina. RESULTS: In the search for therapeutic targets, twenty active components of toad skin were screened for further study, five hundred and sixty-eight targets of components were identified. In the search for disease targets, three thousand two hundred and twenty-seven genes were identified after removal of duplicated genes, one hundred and fifty-nine genes were up-regulated in liver cancer samples while two hundred and seventy-eight were down-regulated in liver cancer patients. After predicting the therapeutic targets of the components, the results were cross-checked with the disease targets, thirteen up-regulated targets and ten down-regulated targets were obtained. Finally, in the results of molecular docking, seven targets (CDK1, AKR1B1, MMP12, AURKB, CHEK1, AURKA, TTK) were potential up-regulated targets, three targets (SHBG, SRD5A2, NR1I2) were potential down-regulated targets, all of which have the best binding energy and molecular interactions. CONCLUSION: CDK1, AKR1B1, MMP12, AURKB, CHEK1, AURKA, and TTK could be potential upregulated target proteins of Huachansu injection for treating liver cancer. The mechanism of Huachansu injection in the treatment of liver cancer through these up-regulated targets is related to cell cycle, cellular senescence, viral carcinogenesis, p53 signaling pathway. SHBG, SRD5A2, and NR1I2 could be potential down-regulated target proteins of Huachansu injection in treating liver cancer.

Clinical efficacy and safety of Huachansu injection combination with platinum-based chemotherapy for advanced non-small cell lung cancer: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Huachansu injection (HCS) is a widely used traditional Chinese medicine for advanced non-small cell lung cancer (NSCLC) to alleviate the adverse drug reactions (ADRs) and enhance the clinical efficacy of chemotherapy. OBJECTIVE: To evaluate the efficacy and safety of HCS as an adjunctive treatment to platinum-based chemotherapy (PBC) for advanced NSCLC. METHODS: A systematic review and meta-analysis were conducted according to PRISMA guidelines. A total of nine databases were searched to select randomized controlled trials (RCTs) of HCS plus PBC to treat NSCLC from inception to October 10, 2020. RCTs on HCS plus PBC vs PBC alone for advanced NSCLC were included. Dichotomous data were pooled as risk ratio (RR) with 95% confidence intervals. RCTs compared to HCS plus PBC vs PBC alone were included. Primary outcomes were objective response rate (ORR) and disease control rate (DCR), and secondary outcomes were survival rate, quality of life (QOL), and adverse drug reactions (ADRs). GRADE software was used to access the quality of evidence. RESULTS: A total of 32 RCTs, including 2753 patients, were included. Compared to PBC alone, HCS plus PBC improved the ORR, DCR, 1- and 2-year survival rates, and QOL and alleviated neutropenia, thrombocytopenia, nausea, vomiting, anemia, liver injury, renal injury, and alopecia. CONCLUSIONS: Compared to PBC alone, HCS plus PBC improved the clinical efficacy and alleviated the ADRs in advanced NSCLC patients. Considering the limitations of the included RCTs, high-quality trials with longer follow-ups are needed to further confirm the results.

Effectiveness of Huachansu injection combined with chemotherapy for treatment of gastric cancer in China: a systematic review and Meta-analysis.

OBJECTIVE: to evaluate the effectiveness and safety of Huachansu (HCS) injection plus chemotherapy in the treatment of gastric cancer. METHODS: A thorough and systematic retrieval of randomized controlled trials (RCTs) concerning HCS injection for treating gastric cancer was conducted in several electronic databases from inception to May 10, 2018. The quality of the RCTs was assessed by the Cochrane risk of bias tool. And the data about objective remission rate, performance status, adverse drug reactions (ADRs) and other outcomes were extracted and analyzed by Review Manager 5.3 and Stata 13.0 software. RESULTS: A total of 14 RCTs with 976 participants were involved in the current Meta-analysis. The results suggested that HCS injection combined with chemotherapy was associated with better effects than receiving conventional chemotherapy alone in respect of improving the objective response rate [RR = 1.18, 95% CI (1.03, 1.37), Z = 2.32, P = 0.02], and performance status [RR = 1.84,95% CI (1.43, 2.36), Z = 4.74, P < 0.000 01]. In addition, HCS injection combined with chemotherapy could relieve pain for patients with gastric cancer. CONCLUSION: This Meta-analysis revealed that HCS injection plus chemotherapy might more effective than chemotherapy in treating gastric cancer. Nevertheless, more large-scale and rigorously designed RCTs should be performed to validate this finding.

Hiponatremia grave por ritual de purificación con veneno de rana amazónica (Kambô) / Severe hyponatremia after a purification ritual using an Amazonian frog poison (Kambô)

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Alpha-7 Nicotinic Receptor-Targeted Cinobufagin Induces Antinociception and Inhibits NF-κB Signaling Pathway in DRG Neurons.

Cinobufagin (CBG) has been shown to have antinociceptive properties. Nevertheless, the antinociceptive effect and mechanism of CBG are still unclear. The present study was designed to investigate the antinociceptive effect of CBG in the thermal and chemical pain models and further to explore the molecular target and potential signal pathway. As shown in the hot-plate test, formalin test, and acetic acid writhing test in mice, administration of CBG produced significant antinociceptive activity in a dose-dependent manner, and the antinociceptive effect was blocked by intraperitoneal pretreatment of methyllycaconitine citrate (an α7 nicotinic receptor antagonist) and intrathecal delivery of an α7 nicotinic receptor antagonist siRNA (α7-siRNA). Immunofluorescence demonstrated that the α7 nicotinic receptor and IκB/NF-κB were coexpressed in primary cultured lumbar DRG neurons. In the chemical pain models and primary cultured DRG neurons, Western blot analysis showed that the formation of p-IκB and p-NF-κB was regulated by CBG, and the effect of CBG was inhibited by α7-siRNA, and ELISA analysis indicated that CBG also regulated the expression of inflammatory cytokines through the α7 nicotinic receptor in DRG. These results suggest that CBG may activate an α7 nicotinic receptor, thereby triggering the inhibition of the DRG NF-κB signaling pathway, resulting in an antinociceptive effect in mice.

Huachansu suppresses TRPV1 up-regulation and spinal astrocyte activation to prevent oxaliplatin-induced peripheral neuropathic pain in rats.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a major dose- and therapy-limiting side effect that is particularly difficult to treat. Huachansu, an aqueous extract from toad skin, is a widely used anti-cancer natural product in China. Clinical findings have established the safety and effectiveness of Huachansu in combination with chemotherapy to promote the therapeutic efficacy while alleviate the side effects, especially cancer-related pain symptoms. Unfortunately, experimental data on the effects and mechanisms of Huachansu in combination with chemotherapy is not available. In this study, the effects of Huachansu were tested in vivo on a rat model of oxaliplatin-induced CIPNP. The results show, a single injection of Huachansu 2.5 g/kg produced a short-term analgesic effect on pre-established oxaliplatin-induced CIPNP after 60 min, as indicated by decreased mechanical and thermal hypersensitivity in comparison to oxaliplatin-treated rats. Repeated doses of Huachansu, given during CIPNP induction, prevented the development of oxaliplatin-induced CIPNP. This prophylactic effect of Huachansu was associated with suppressed oxaliplatin-induced TRPV1 up-regulation in the dorsal root ganglia and spinal astrocyte activation. These findings reveal Huachansu therapeutic potential in treating and preventing CIPNP.

The syndrome of inappropriate antidiuretic hormone secretion after giant leaf frog (Phyllomedusa bicolor) venom exposure.

INTRODUCTION: In Europe body purification and natural balance restoring rituals are becoming increasingly popular, but an introduction of Amazonian shamanic rituals in urban Europe can result in unexpected adverse events. CASE REPORT: A 44-year-old woman attended a Kambô or Sapo ritual in Slovenia where dried skin secretion from a giant leaf frog (Phyllomedusa bicolor) was applied to five freshly burned wounds at her shoulder. Afterwards, she drank 6 litres of water and gradually developed nausea and vomiting, confusion, lethargy, muscle weakness, spasms and cramps, seizure, decreased consciousness level and short-term memory loss. The initial laboratory tests showed profound plasma hypoosmolality (251 mOsm/kg) proportional to hyponatremia (116 mmol/L) combined with inappropriately elevated urine osmolality (523 mOsm/kg) and high urine sodium concentration (87 mmol/L) indicating a syndrome of inappropriate antidiuretic hormone secretion. The patient was treated with 0.9% sodium chloride and a restriction of water intake. Plasma osmolality and hyponatremia improved one day after venom exposure, but the symptoms disappeared as late as the third day. CONCLUSION: In patients presenting with neurological symptoms and a line of small body burns Phyllomedusa bicolor venom exposure should be suspected. Acute symptomatic hyponatremia after Phyllomedusa bicolor venom exposure is the result of inappropriate antidiuretic hormone secretion that can be exacerbated by excessive water intake.

Effects of cinobufacini injection on cell proliferation and the expression of topoisomerases in human HepG-2 hepatocellular carcinoma cells.

The present study aimed to investigate the effects of cinobufacini injection on the proliferation and expression of topoisomerases in human HepG-2 hepatocarcinoma cells. The cells were divided into a control group and an experimental group, in which 0.105, 0.21, 0.42 mg/l cinobufacini was injected. Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay, levels of apoptosis were detected using annexin V/propidium iodide staining and cell cycles were analyzed using flow cytometric analysis. The mRNA and protein expression levels of topoisomerase (TOPO) I and TOPO II were determined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Cinobufacini injection significantly inhibited the proliferation of the HepG-2 cells (P<0.05), induced apoptosis (P<0.05) in a dose- and time-dependent manner, induced tumor cell arrest at the S phase in a dose-dependent manner, and downregulated the mRNA and protein expression levels of TOPO I and TOPO II (P<0.05) in a dose-dependent manner. Therefore, cinobufacini was found to inhibit human HepG-2 hepatocellular carcinoma cell proliferation, and downregulation of the expression levels of TOPO I and TOPO II may contribute to the effect on proliferation observed in the Hep­G2 cells following cinobbufacini injection.

A meta-analysis of Cinobufacini combined with transcatheterarterial chemoembolization in the treatment of advanced hepatocellular carcinoma.

OBJECTIVE: The aim of this meta-analysis is to evaluate the clinical efficacy of cinobufacini combined with transcatheter arterial chemoembolization (TACE) in the treatment of advanced hepatocellular carcinoma. MATERIALS AND METHODS: By searching Medline, the Cochrane central register of controlled trials, EMBSE, ESMO, Wanfang and Chinese National Knowledge Infrastructure (CNKI) databases, the open published clinical trials compared the clinical efficacy of cinobufacini plus TACE versus TACE only in patients with advanced hepatocellular carcinoma were collected. The pooled objective response rate (ORR) and 1 or 2 year survival rate were calculated by Stata11.0 statistical software. RESULTS: Nine studies including a total of 659 subjects (333 in cinobufacini plus TACE and 326 in TACE only) were finally included in this meta-analysis. The pooled analysis demonstrated that cinobufacini plus TACE can significant increase the objective response rate (ORR) compared with TACE only with an relative risk of 1.28 (P = 0.006); The 1-year survival rate in cinobufacini plus TACE group was not significant difference compared with TACE only by pooling the data (RR = 1.24, P = 0.13); But the 2-year survival rate in cinobufacini plus TACE group was much higher than that of TACE only group with an RR of 2.0, (P = 0.001). CONCLUSION: This meta-analysis demonstrated that cinobufacini combined with TACE can significantly increase the objective response rate and 2-year survival rate compared with TACE only in patients with advanced hepatocellular carcinoma.

A case of advanced lung cancer with malignant pericardial effusion treated by intrapericardial Cinobufacini injection instillation.

Malignant pericardial effusion is one of the severe complications in advanced lung cancer patients, seriously affecting the patient's cardiopulmonary function and even life. Pericardial drainage and instillation of anti-neoplastic drugs in the pericardial cavity seems to offer the best chance of controlling pericardial effusion. We reported a case concerning treatment of a 63-year-old man in advanced lung cancer with a large amount of pericardial effusion. We utilized pericardium puncture and drainage combined with instillation of Cinobufacini injection in the pericardial cavity to treat pericardial effusion. After treatment with Cinobufacini injection for two weeks, the patient was followed up in one month to assess effectiveness, quality of life, and safety. We found that the cardiac tamponade symptoms such as difficult breathing, chest distress, and palpitations were significantly relieved. The patient's quality of life was effectively improved with KPS scores increased. We also found that the levels of tumor marker CA-125 in the pericardial effusion decreased (from 340.80 U/mL to 34.85 U/mL) and pericardium B ultrasound showed that the quantity of pericardial effusion reduced significantly (from 2.5 cm to 0.6 cm). Furthermore, there were little gastrointestinal adverse reactions and myelosuppression in the patient after instillation of the Cinobufacini injection. Taken together, this provides a new way for treating cancerous pericardial effusion, especially for patients who cannot tolerate instillation of chemotherapy drugs, and is worthwhile to carry out more standardized studies in the future.

Preparation and in vitro anti-tumor properties of toad venom extract-loaded solid lipid nanoparticles.

In this study, we prepared solid lipid nanoparticles (TV-SLNs) loaded with toad venom extract and investigated their anti-tumor effects in vitro in HeLa and SKOV-3 cells. TV-SLNs were prepared using a cold homogenization technique, and the formulation was optimized by central composite design and response surface methods. The anti-tumor activities of TV-SLNs were evaluated by analyzing cell division and cell cycle distribution by using the MTT assay and flow cytometry. After incubation with TV-SLNs, the growth of both HeLa and SKOV-3 cells was inhibited significantly. The percentage of HeLa cells in G0/G1 phase decreased, whereas that in the S and G2/M phases increased. Thus, the S and G2/M phases were blocked after the incubation of HeLa cells with TV-SLNs for 24 h. In contrast, the percentage of SKOV-3 cells in G0/G1 phase increased and then decreased in S and G2/M phases, with the G0/G1 phase being blocked after incubation with TV-SLNs for 24 h. Our results demonstrate that TV-SLNs inhibited the fissiparism of HeLa and SKOV-3 cells in a time-and dose-dependent manner. TV-SLNs may be effective as a novel TV vaginal delivery system for the treatment of cervical and ovarian cancers.

[In vitro anti-proliferation effect of peptides from cinobufacini injection].

Cinobufacini is an aqueous extract of Bufo bufo gargarizans Cantor dried skin, which has been widely used for cancer therapy in China. So far, its active components are still not very clear. In previous reports, bufadienolides with low-concentration were usually studied because of their anticancer effects. However, the high polarity constituents in cinobufacini are less investigated. The present study found that more than 50% contents of cinobufacini were water-soluble peptides. Then, in vitro anticancer experiments were carried out, including human stomach cancer cell lines BGC823 and MCG803, human colon cancer cell lines DLD-1 and HT-29, and human pancreatic cancer cell line MIAPACA-2. The IC50 for these cell lines model were ranged from 25-123 microgmL(-1). The results indicated that these peptides showed similar activity with cinobufacini injection. As a conclusion, this study provides a new and further understanding of anticancer components in cinobufacini injection.

Prospective randomised evaluation of traditional Chinese medicine combined with chemotherapy: a randomised phase II study of wild toad extract plus gemcitabine in patients with advanced pancreatic adenocarcinomas.

BACKGROUND: An intravenous formulated extract of the venom of the wild toad Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, huachansu, is currently used in China for the treatment of lung, liver, pancreatic, and colorectal cancers. We performed a randomised, single-blinded, phase II clinical study of huachansu plus gemcitabine versus placebo plus gemcitabine in patients with locally advanced and/or metastatic pancreatic adenocarcinomas. METHODS: Patients with tissue-proven locally advanced and/or metastatic pancreatic adenocarinoma were randomly assigned to receive either gemcitabine 1000 mg m(-2) on days 1, 8, and 15 with huachansu 20 ml m(-2) daily for 21 days (arm A) or placebo (arm B); treatment cycles were 28 days in length. Primary end point was 4-month progression-free overall survival (PFS); secondary end points were objective radiographical response rate (ORR), time to progression (TTP), and toxicity. RESULTS: A total of 80 subjects were enrolled; 76 patients were evaluable (received at least 1 week therapy). Median overall survival was 160 days for arm A and 156 days for arm B (P=0.339); ORR was 9 and 3% in arms A and B, respectively (P=0.332), median TTP was 98 and 115 days, respectively (P=0.825); the median 4-month PFS was 99 and 98 days, respectively (P=0.679). CONCLUSION: Huachansu when combined with gemcitabine did not improve the outcome of patients with locally advanced and/or metastatic pancreatic cancer.

Preventive effects of jiedu granules combined with cinobufacini injection versus transcatheter arterial chemoembolization in post-surgical patients with hepatocellular carcinoma: a case-control trial.

OBJECTIVE: To investigate the therapeutic effects of Jiedu granules, a Chinese medicine (CM) compound, plus cinobufacini injection, which was extracted from skin of Bufo bufo gargarizans Cantor, to prevent the recurrence of hepatocellular carcinoma (HCC) after surgical resection. METHODS: In this case-control trial, a total of 120 patients who stayed in Changhai Hospital were enrolled from December 2001 to December 2006. Sixty patients were treated with Jiedu granules plus cinobufacini injection to prevent tumor recurrence after operation (CM group) and 60 patients were treated with transcatheter arterial chemoembolization (TACE) after operation (TACE group). Progression-free survival (PFS) and overall survival (OS) rates were determined to evaluate the therapeutic effects of post-operative management of patients with HCC. RESULTS: PFS in the CM group was 18.07 months [95% confidence interval (CI): 12.49-23.65] and the 1-, 2-, 3-, 4- and 5-year PFS rates were 61%, 39%, 26%, 22% and 12%, respectively. PFS in the TACE group was 8.03 months (95% CI: 6.63-9.44) and the 1-, 2-, 3-, 4- and 5-year PFS rates were 34%, 11%, 7%, 2% and 0%, respectively. There was significant difference in survival rate between the two groups (P<0.01). The mean survival time (MST) of patients in the CM group was 49.53 months versus 39.90 months of the TACE group. The 1-, 2-, 3-, 4- and 5-year survival rates were 90%, 82%, 80%, 70% and 63%, respectively, in the CM group, and 79%, 70%, 60%, 60% and 36%, respectively, in the TACE group. There was significant difference in survival time between the two groups (P=0.045). CONCLUSIONS: Jiedu granules plus cinobufacini injection, a combination that is commonly used for post-operation management of HCC, can postpone tumor recurrence and metastasis, prolong the survival time and increase the survival rate of post-surgical patients with HCC. However, these findings need to be confirmed in a prospective, randomized controlled trial.

Bufothionine, a possible effective component in cinobufocini injection for hepatocellular carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini has been traditionally used in China for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer. MATERIALS AND METHODS: High performance liquid chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles. RESULTS: HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 µg/ml). CONCLUSIONS: These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.

Antitumor effect of skin of venenum bufonis in a NCI-H460 tumor regression model.

This experimental study was performed to investigate the antitumor effect of skin of venenum bufonis (SVB) in NCI-H460 human lung cancer cell xenografted nude mice. NCI-H460 cell lines were cultured and then xenografted into nude mice. Mice were divided into four groups: SVB (0.25 mg/kg) given orally, SVB (0.25 mg/kg) interperitoneally, SVB (0.5 mg/kg) interperitoneally, and the untreated group. Mice were raised and treated for 28 days. Body weight and tumor weight and volumes were measured daily. Absolute organ weight, microhistological observations and biochemical blood analyses were performed on the final day of the study following the sacrificing of these animals. Tumor inhibition rate, mean survival time and percent increase in life span were also calculated. Tumor size decreased in all SVB treated mice. Increasing the dose of SVB attenuated the inhibition rate seen on the 11th day of this experiment. Furthermore, tumor weight and volume in the mice treated with the highest dose of SVB were the smallest. Mice treated with high-dose intraperitoneal SVB gained weight and had significantly smaller spleens compared with untreated mice. Mean survival time and percent increase in life span in the low-dose intraperitoneal SVB treatment group were higher than those of other groups. Biochemical blood analysis revealed that phosphatase and urea nitrogen levels were decreased significantly in 0.25 mg/kg SVB orally treated mice (p < 0.01). Blood level calcium and alanine transaminase significantly decreased with intraperitoneal SVB 0.5 mg/kg (p < 0.05). The findings of this in vivo study suggest that SVB may have potential as a tumor growth inhibitor. Further research that overcomes the limitations of this study to determine the antitumor mechanism of SVB is still required.

Pilot study of huachansu in patients with hepatocellular carcinoma, nonsmall-cell lung cancer, or pancreatic cancer.

BACKGROUND: Huachansu, a Chinese medicine that comes from dried toad venom from the skin glands of Bufo gargarizans or B. melanostictus, has been used in the treatment of various cancers in China. The authors conducted a pilot study, using a phase 1 trial design, of huachansu in patients with advanced cancer. METHODS: Huachansu was administered intravenously for 14 days followed by 7 days off (1 cycle). Without significant adverse events or progressive disease, treatment continued beyond 2 cycles. The dose of huachansu was escalated as follows with 3 patients per cohort: 10 (level 1), 20 (level 2), 40 (level 3), 60 (level 4), and 90 (level 5) mL/m(2). RESULTS: Fifteen patients (hepatocellular cancer, n = 11; nonsmall cell lung cancer, n = 2; pancreatic cancer, n = 2) were enrolled in the trial, and no dose-limiting toxicities (DLTs) were found. Eleven patients had no drug-related toxicity greater than grade 1. Six (40%) had stable disease (median duration, 6.0 months; range, 3.5-11.1 months). One of these patients (with hepatocellular cancer) had 20% regression (duration, 11 months) (dose level 1). Quality of life improved for patients with stable disease. Plasma bufalin concentration reached maximal levels at the end of the 2-hour infusion and was proportional to the amount of drug being administered (0.81-3.38 ng/mL). CONCLUSIONS: No DLT was observed with the use of huachansu at doses up to 8x higher than typically used in China. Six patients had prolonged stable disease or minor tumor shrinkage.