Degradation of MCL-1 by bufalin reverses acquired resistance to osimertinib in EGFR-mutant lung cancer.

Although osimertinib, an EGFR tyrosine kinase inhibitor, has become the standard therapy for treating non-small cell lung cancer (NSCLC) patients with EGFR-activating mutation, upregulation of MCL-1 induces acquired resistance to osimertinib. Bufalin, a natural digoxin-like ingredient isolated from a traditional Chinese medicine Chan Su, has been shown to downregulate MCL-1 in NSCLC cells. However, whether bufalin reverses this acquired resistance to osimertinib in NSCLC cells remains unclear. In this study, bufalin reduced cell viability and promoted apoptosis in osimertinib-resistant cells. Moreover, co-treatment with bufalin and osimertinib restored the sensitivity of osimertinib-resistant cells to osimertinib-induced growth regression and apoptosis in vitro and in vivo. Mechanistically, MEK/ERK-dependent MCL-1 phosphorylation and Ku70-mediated MCL-1 overexpression confer osimertinib resistance in EGFR-mutant NSCLC cells. In osimertinib-resistant cells, bufalin modulates Ku70-mediated MCL-1 degradation, but not MEK/ERK/MCL-1 signaling. In conclusion, our study suggests that bufalin eliminates resistance to osimertinib by inhibiting Ku70-mediated MCL-1 overexpression, indicating that a combination of osimertinib and bufalin could be an effective additional treatment to overcome acquired resistance to osimertinib in NSCLC cells.

Degradation of Mcl-1 through GSK-3ß Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells.

BACKGROUND/AIMS: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. METHODS: Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. RESULTS: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3ß. GSK-3 inhibitor and GSK-3ß siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. CONCLUSION: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3ß activation was involved in bufalin-induced apoptosis.

[Tumor microenvironment elicits primary resistance to afatinib through HGF secretion].

OBJECTIVE: To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib. METHODS: The effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib. RESULTS: The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin. CONCLUSIONS: HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

[Effect of bufalin combined gefitinib on lung cancer H1975 cells and its mechanisms research].

OBJECTIVE: To observe the effect of bufalin combined Gefitinib on lung cancer H1975 cells, and to explore its potential mechanisms for anti-tumor. METHODS: The cytostatic effects of bufalin (1 -100 nmol/L), gefitinib (0.1-20 micromol/L), and bufalin plus gefitinib on H1975 cells were evaluated by MTT assay. Their effects on apoptosis of H1975 cells were determined by flow cytometry (FCM). Their effects on expressions of epidermal growth factor receptor (EGFR) and Met signal pathway related proteins in H1975 cells were detected by Western blot. RESULTS: Results of MTT assay showed that gefitinib over 5 micromol/L could inhibit H1975 cells. But combined therapy of bufalin and gefitinib could potently inhibit the growth of H1975 cells. Results of FCM showed the apoptotic rate was 61.64% +/- 5.61% in the bufalin plus gefitinib group, obviously higher than that of the bufalin group (18.34% +/- 3.42%) and the gefitinib group (7.32% +/- 1.08%), showing statistical difference (P < 0.01). Results of Western blot showed the protein expressions of p-EGFR, p-Met, p-Akt, and p-mTOR in H1975 cells could be markedly down-regulated by bufalin plus gefitinib. CONCLUSIONS: Combination of bufalin and gefitinib potently inhibited the growth of H1975 cells, and induced cell apoptosis. The potential mechanism for anti-tumor might be involved in blocking EGFR-PI3k/Akt pathway.

Nomogram for predicting the preoperative lymph node metastasis in resectable pancreatic cancer.

BACKGROUND: Lymph node metastasis (LNM) is a critical prognostic factor in resectable pancreatic cancer (PC) patients, determining treatment strategies. This study aimed to develop a clinical model to adequately and accurately predict the risk of LNM in PC patients. METHODS: 13,200 resectable PC patients were enrolled from the SEER (Surveillance, Epidemiology, and End Results) database, and randomly divided into a training group and an internal validation group at a ratio of 7:3. An independent group (n = 62) obtained from The First Affiliated Hospital of Xinxiang Medical University was enrolled as the external validation group. The univariate and multivariate logistic regression analyses were used to screen independent risk factors for LNM. The minimum Akaike's information criterion (AIC) was performed to select the optimal model parameters and construct a nomogram for assessing the risk of LNM. The performance of the nomogram was assessed by the receiver operating characteristics (ROC) curve, calibration plot, and decision curve analysis (DCA). In addition, an online web calculator was designed to assess the risk of LNM. RESULT: A total of six risk predictors (including age at diagnosis, race, primary site, grade, histology, and T-stage) were identified and included in the nomogram. The areas under the curves (AUCs) [95% confidential interval (CI)] were 0.711 (95%CI: 0.700-0.722), 0.700 (95%CI: 0.683-0.717), and 0.845 (95%CI: 0.749-0.942) in the training, internal validation and external validation groups, respectively. The calibration curves showed satisfied consistency between nomogram-predicted LNM and actual observed LNM. The concordance indexes (C-indexes) in the training, internal, and external validation sets were 0.689, 0.686, and 0.752, respectively. The DCA curves of the nomogram demonstrated good clinical utility. CONCLUSION: We constructed a nomogram model for predicting LNM in pancreatic cancer patients, which may help oncologists and surgeons to choose more individualized clinical treatment strategies and make better clinical decisions.

Nomograms for predicting overall survival and cancer-specific survival in elderly patients with epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is one of the most fatal gynecological malignancies among elderly patients. We aim to construct two nomograms to predict the overall survival (OS) and cancer-specific survival (CSS) in elderly EOC patients. METHODS: Elderly patients with EOC between 2000 and 2019 were selected from the Surveillance, Epidemiology, and End Results (SEER) database. Enrolled patients were randomly divided into the training and validation set at a ratio of 2:1. The OS and CSS were recognized as endpoint times. The independent prognostic factors from the multivariate analysis were used to establish nomograms for predicting the 3-, 5- and 10-year OS and CSS of elderly EOC patients. The improvement of predictive ability and clinical benefits were evaluated by consistency index (C-index), receiver operating characteristic (ROC), calibration curve, decision curve (DCA), net reclassification improvement (NRI), and integrated discrimination improvement (IDI). Finally, the treatment efficacy of surgery and chemotherapy in low-, medium-, and high-risk groups were displayed by Kaplan-Meier curves. RESULTS: Five thousand five hundred eighty-eight elderly EOC patients were obtained and randomly assigned to the training set (n = 3724) and validation set (n = 1864). The independent prognostic factors were utilized to construct nomograms for OS and CSS. Dynamic nomograms were also developed. The C-index of the OS nomogram and CSS nomogram were 0.713 and 0.729 in the training cohort. In the validation cohort, the C-index of the OS nomogram and CSS nomogram were 0.751 and 0.702. The calibration curve demonstrated good concordance between the predicted survival rates and actual observations. Moreover, the NRI, IDI, and DCA curves determined the outperformance of the nomogram compared with the AJCC stage system. Besides, local tumor resection had a higher benefit on the prognosis in all patients. Chemotherapy had a better prognosis in the high-risk groups, but not for the medium- risk and low-risk groups. CONCLUSIONS: We developed and validated nomograms for predicting OS and CSS in elderly EOC patients to help gynecologists to develop an appropriate individualized therapeutic schedule.

The chemical composition of human milk from Inner Mongolia of China.

In Inner Mongolia, China, the chemical composition of 66 breast milk samples at three lactation stages was analysed. Except for total nitrogen content, the contents of total solid, fat, NPN, lactose and ash were not significantly different between colostral, transitional and mature milk. Fatty acids did not vary over the three lactation stages, while unsaturated fatty acids accounted for 59.95-63.22% of the total fatty acids. Relatively low contents of vitamins were in the milk because the volunteer mothers did not take any vitamin supplementation over the entire lactation period. Besides sodium and phosphate, the concentrations of most minerals in the breast milk remained fairly constant across the three lactation stages.

Bufalin Reverses HGF-Induced Resistance to EGFR-TKIs in EGFR Mutant Lung Cancer Cells via Blockage of Met/PI3k/Akt Pathway and Induction of Apoptosis.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have shown promising therapeutic efficacy in nonsmall cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor- (EGFR-) activating mutation. However, the inevitable recurrence resulting from acquired resistance has limited the clinical improvement in therapy outcomes. Many studies demonstrate that hepatocyte growth factor- (HGF-) Met axis plays an important role in tumor progression and drug sensitivity. HGF may induce resistance to EGFR-TKIs in EGFR mutant lung cancer cells by Met/PI3K/Akt signaling. The purpose of this study was to determine whether bufalin, a major bioactive component of Venenum Bufonis, could reverse HGF-induced resistance to reversible and irreversible EGFR-TKIs in mutant lung cancer cells PC-9, HCC827, and H1975. Our studies showed that bufalin could reverse resistance to reversible and irreversible EGFR-TKIs induced by exogenous HGF in EGFR mutant lung cancer cells by inhibiting the Met/PI3K/Akt pathway and inducing death signaling. These results suggested that bufalin might have a potential to overcome HGF-induced resistance to molecular-targeted drugs for lung cancer.