Inhibitory effects of everolimus in combination with paclitaxel on adriamycin-resistant breast cancer cell line MDA-MB-231.

OBJECTIVE: We aimed to evaluate the therapeutic effects of paclitaxel in combination with mTOR inhibitor everolimus on adriamycin-resistant breast cancer cell line MDA-MB-231 (MDA-MB-231/ADR). MATERIALS AND METHODS: MDA-MB-231/ADR cells were treated with different concentrations of paclitaxel and everolimus. The IC50 values after 48 h of treatment were measured by the MTT assay. The apoptosis rate and cell cycle were detected by flow cytometry. The protein expressions of Akt, PI3K, mTOR, p-pI3K, p-AKT and p-mTOR were detected by Western blot. RESULTS: When paclitaxel at ≥1.56 µg/ml was used, the growth of MDA-MB-231/ADR cells was inhibited more significantly than that of control group (P < 0.05). After treatment with ≥6.25 µg/ml everolimus, the cell growth was also suppressed more significantly (P < 0.05). The IC50 values of everolimus and paclitaxel were 32.50 µg/ml and 7.80 µg/ml, respectively. The inhibition rate of paclitaxel plus everolimus was significantly enhanced with increasing paclitaxel concentration (P < 0.001). After treatment with 7.80 µg/ml paclitaxel, the two drugs had best synergistic inhibitory effects on proliferation. Compared with drugs alone, the combination significantly promoted apoptosis (P < 0.001). The paclitaxel + everolimus group had significantly more cells in the G0-G1 phase than those of control and individual drug groups (P < 0.001). Everolimus significantly decreased mTOR and p-mTOR expressions compared with those of control group (P < 0.001). Compared with everolimus alone, the combination reduced the expressions more significantly (P < 0.05). Paclitaxel decreased the expression levels of PI3K, p-PI3K and p-AKT. Compared with paclitaxel alone, the combination significantly promoted the reduction of PI3K, p-PI3K and p-AKT expressions (P < 0.05). CONCLUSION: Everolimus can enhance the effect of paclitaxel on MDA-MB-231/ADR cells, inhibit cell proliferation, induce apoptosis and arrest cell cycle in the G1 phase mainly by down-regulating the expressions of key proteins in the mTOR signaling pathway.

Caveolin-1 inhibits breast cancer stem cells via c-Myc-mediated metabolic reprogramming.

Breast cancer stem cells (BCSCs) are considered to be the root of breast cancer occurrence and progression. However, the characteristics and regulatory mechanisms of BCSCs metabolism have been poorly revealed, which hinders the development of metabolism-targeted treatment strategies for BCSCs elimination. Herein, we demonstrated that the downregulation of Caveolin-1 (Cav-1) usually occurred in BCSCs and was associated with a metabolic switch from mitochondrial respiration to aerobic glycolysis. Meanwhile, Cav-1 could inhibit the self-renewal capacity and aerobic glycolysis activity of BCSCs. Furthermore, Cav-1 loss was associated with accelerated mammary-ductal hyperplasia and mammary-tumor formation in transgenic mice, which was accompanied by enrichment and enhanced aerobic glycolysis activity of BCSCs. Mechanistically, Cav-1 could promote Von Hippel-Lindau (VHL)-mediated ubiquitination and degradation of c-Myc in BCSCs through the proteasome pathway. Notably, epithelial Cav-1 expression significantly correlated with a better overall survival and delayed onset age of breast cancer patients. Together, our work uncovers the characteristics and regulatory mechanisms of BCSCs metabolism and highlights Cav-1-targeted treatments as a promising strategy for BCSCs elimination.

Adjuvant Capecitabine With Docetaxel and Cyclophosphamide Plus Epirubicin for Triple-Negative Breast Cancer (CBCSG010): An Open-Label, Randomized, Multicenter, Phase III Trial.

PURPOSE: Standard adjuvant chemotherapy for triple-negative breast cancer (TNBC) includes a taxane and an anthracycline. Concomitant capecitabine may be beneficial, but robust data to support this are lacking. The efficacy and safety of the addition of capecitabine into the TNBC adjuvant treatment regimen was evaluated. PATIENTS AND METHODS: This randomized, open-label, phase III trial was conducted in China. Eligible female patients with early TNBC after definitive surgery were randomly assigned (1:1) to either capecitabine (3 cycles of capecitabine and docetaxel followed by 3 cycles of capecitabine, epirubicin, and cyclophosphamide) or control treatment (3 cycles of docetaxel followed by 3 cycles of fluorouracil, epirubicin, and cyclophosphamide). Randomization was centralized without stratification. The primary end point was disease-free survival (DFS). RESULTS: Between June 2012 and December 2013, 636 patients with TNBC were screened, and 585 were randomly assigned to treatment (control, 288; capecitabine, 297). Median follow-up was 67 months. The 5-year DFS rate was higher for capecitabine than for control treatment (86.3% v 80.4%; hazard ratio, 0.66; 95% CI, 0.44 to 0.99; P = .044). Five-year overall survival rates were numerically higher but not significantly improved (capecitabine, 93.3%; control, 90.7%). Overall, 39.1% of patients had capecitabine dose reductions, and 8.4% reported grade ≥ 3 hand-foot syndrome. The most common grade ≥ 3 hematologic toxicities were neutropenia (capecitabine, 136 [45.8%]; control, 118 [41.0%]) and febrile neutropenia (capecitabine, 50 [16.8%]; control, 46 [16.0%]). Safety data were similar to the known capecitabine safety profile and generally comparable between arms. CONCLUSION: Capecitabine when added to 3 cycles of docetaxel followed by 3 cycles of a 3-drug anthracycline combination containing capecitabine instead of fluorouracil significantly improved DFS in TNBC without new safety concerns.

Betulinic Acid Suppresses Breast Cancer Metastasis by Targeting GRP78-Mediated Glycolysis and ER Stress Apoptotic Pathway.

Targeting aberrant metabolism is a promising strategy for inhibiting cancer growth and metastasis. Research is now geared towards investigating the inhibition of glycolysis for anticancer drug development. Betulinic acid (BA) has demonstrated potent anticancer activities in multiple malignancies. However, its regulatory effects on glycolysis and the underlying molecular mechanisms are still unclear. BA inhibited invasion and migration of highly aggressive breast cancer cells. Moreover, BA could suppress aerobic glycolysis of breast cancer cells presenting as a reduction of lactate production, quiescent energy phenotype transition, and downregulation of aerobic glycolysis-related proteins. In this study, glucose-regulated protein 78 (GRP78) was also identified as the molecular target of BA in inhibiting aerobic glycolysis. BA treatment led to GRP78 overexpression, and GRP78 knockdown abrogated the inhibitory effect of BA on glycolysis. Further studies demonstrated that overexpressed GRP78 activated the endoplasmic reticulum (ER) stress sensor PERK. Subsequent phosphorylation of eIF2α led to the inhibition of ß-catenin expression, which resulted in the inhibition of c-Myc-mediated glycolysis. Coimmunoprecipitation assay revealed that BA interrupted the binding between GRP78 and PERK, thereby initiating the glycolysis inhibition cascade. Finally, the lung colonization model validated that BA inhibited breast cancer metastasis in vivo, as well as suppressed the expression of aerobic glycolysis-related proteins. In conclusion, our study not only provided a promising drug for aerobic glycolysis inhibition but also revealed that GRP78 is a novel molecular link between glycolytic metabolism and ER stress during tumor metastasis.

Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage.

Accumulating evidence suggests that caveolin-1 (CAV-1) is a stress-related oncotarget and closely correlated to chemoresistance. Targeting CAV-1 might be a promising strategy to improve chemosensitivity for breast cancer treatment. Astragaloside IV (AS-IV), a bioactive compound purified from Astragalus membranaceus, has been shown to exhibit multiple bioactivities, including anticancer. However, the involved molecular targets are still ambiguous. In this study, we investigated the critical role of CAV-1 in mediating the chemosensitizing effects of AS-IV to Taxol on breast cancer. We found that AS-IV could enhance the chemosensitivity of Taxol with minimal direct cytotoxicity on breast cancer cell lines MCF-7 and MDA-MB-231, as well as the nontumor mammary epithelial cell line MCF-10A. AS-IV was further demonstrated to aggravate Taxol-induced apoptosis and G2/M checkpoint arrest. The phosphorylation of mitogen-activated protein kinase (MAPK) signaling extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK), except p38, was also abrogated by a synergistic interaction between AS-IV and Taxol. Moreover, AS-IV inhibited CAV-1 expression in a dose-dependent manner and reversed CAV-1 upregulation induced by Taxol administration. Mechanism study further demonstrated that AS-IV treatment triggered the eNOS/NO/ONOO- pathway via inhibiting CAV-1, which led to intense oxidant damage. CAV-1 overexpression abolished the chemosensitizing effects of AS-IV to Taxol by inhibiting oxidative stress. In vivo experiments further validated that AS-IV increased Taxol chemosensitivity on breast cancer via inhibiting CAV-1 expression, followed by activation of the eNOS/NO/ONOO- pathway. Taken together, our findings not only suggested the potential of AS-IV as a promising candidate to enhance chemosensitivity, but also highlighted the significance of CAV-1 as the target to reverse cancer drug resistance.

Broadleaf Mahonia attenuates granulomatous lobular mastitis­associated inflammation by inhibiting CCL­5 expression in macrophages.

Granulomatous lobular mastitis (GLM) is a type of chronic mammary inflammation with unclear etiology. Currently systematic corticosteroids and methitrexate are considered as the main drugs for GLM treatment, but a high toxicity and risk of recurrence greatly limit their application. It is therefore an urgent requirement that safe and efficient natural drugs are found to improve the GLM prognosis. Broadleaf Mahonia (BM) is a traditional Chinese herb that is believed to have anti­inflammatory properties according to ancient records of traditional Chinese medicine. The present study investigated this belief and demonstrated that BM significantly inhibited the expression of interleukin­1ß (IL­1ß), IL­6, cyclooxygenase­2 and inducible nitric oxide synthase in RAW264.7 cells, but had little influence on the cell viability, cell cycle and apoptosis. Meanwhile, the lipopolysaccharide­induced elevation of reactive oxygen species and nitric oxide was also blocked following BM treatment, accompanied with decreased activity of nuclear factor­κB and MAPK signaling. A cytokine array further validated that BM exhibited significant inhibitory effects on several chemoattractants, including chemokine (C­C motif) ligand (CCL)­2, CCL­3, CCL­5 and secreted tumor necrosis factor receptor 1, among which CCL­5 exhibited the highest inhibition ratio in cell and clinical GLM specimens. Collectively, the results show that BM is a novel effective anti­inflammatory herb in vitro and ex vivo, and that CCL­5 may be closely associated with GLM pathogenesis.

AMPK and Cancer.

This chapter focuses on the role of AMPK as a stress-response molecule with an emphasis on its duplex implication in carcinogenesis and cancer drug resistance. AMPK is closely correlated to the tumor-suppressive functions of LKB1 and P53, consequently modulating the activity of cellular survival signaling such as mTOR and Akt, leading to cell growth inhibition and cell cycle arrest. On the contrary, AMPK is tightly involved in cancer drug resistance via interacting with multiple known mechanisms of chemoresistance such as ABCG2 expression, autophagy induction, and cancer stem cells enrichment. Targeting AMPK has become a novel strategy for cancer prevention and treatment.

The inflammasome: an emerging therapeutic oncotarget for cancer prevention.

Deregulated inflammation is considered to be one of the hallmarks of cancer initiation and development regulation. Emerging evidence indicates that the inflammasome plays a central role in regulating immune cells and cytokines related to cancer. The inflammasome is a multimeric complex consisting of Nod-like receptors (NLRs) and responds to a variety of endogenous (damage-associated molecular patterns) and exogenous (pathogen-associated molecular patterns) stimuli. Several lines of evidence suggests that in cancer the inflammasome is positively associated with characteristics such as elevated levels of IL-1ß and IL-18, activation of NF-κB signaling, enhanced mitochondrial oxidative stress, and activation of autophagic process. A number of NLRs, such as NLRP3 and NLRC4 are also highlighted in carcinogenesis and closely correlate to chemoresponse and prognosis. Although conflicting evidence suggested the duplex role of inflammasome in cancer development, the phenomenon might be attributed to NLRs difference, cell and tissue type, cancer stage, and specific experimental conditions. Given the promising role of inflammasome in mediating cancer development, precise elucidation of its signaling network and pathological significance may lead to novel therapeutic options for malignancy therapy and prevention.

Targeting AMPK Signaling Pathway to Overcome Drug Resistance for Cancer Therapy.

Mulitdrug resistance (MDR) is one of critical factorslimiting the efficacy of cancer chemoor radiotherapy. Emerging evidence has indicated that MDR is a complex process regulated by multiple factors, among which stress response molecules are considered as central players. AMP-activated protein kinase (AMPK) is a major regulator balancing energy supply and ultimately protects cells from harmful stresses via coordinating multiple metabolic pathways Notably, AMPK activation was recently shown to mediate the metabolism reprogramming in drug resistant cancer cells including promoting Warburg effects and mitochondrial biogenesis. Furthermore, AMPK activity has also been shown to regulate the self-renewal ability of cancer stem cells that are often refractory to chemotherapy. In addition, AMPK phosphorylation was critical in mediating autophagy induction, a process demonstrated to be effective in chemosensitivity modulation via degrading cellular components to satisfy nutrients requirement under stressful condition. Meanwhile, drug discovery targeting AMPK has been developed to validate the pathological significance of AMPK in cancer prevention and treatment. Although conflicting evidence focusing on the AMPK modulation for cancer treatment is still remained, this might be attributed to differences in AMPK isotypes in specific tissues, off-targets effects, the degree and duration of drug administration and experimental setting of stress conditions. This review will focus on AMPK mediated resistance to cancer therapy and discuss its potential therapeutic implication and targeting drug development.

Caveolin-1, a stress-related oncotarget, in drug resistance.

Caveolin-1 (Cav-1) is both a tumor suppressor and an oncoprotein. Cav-1 overexpression was frequently confirmed in advanced cancer stages and positively associated with ABC transporters, cancer stem cell populations, aerobic glycolysis activity and autophagy. Cav-1 was tied to various stresses including radiotherapy, fluid shear and oxidative stresses and ultraviolet exposure, and interacted with stress signals such as AMP-activated protein kinase. Finally, a Cav-1 fluctuation model during cancer development is provided and Cav-1 is suggested to be a stress signal and cytoprotective. Loss of Cav-1 may increase susceptibility to oncogenic events. However, research to explore the underlying molecular network between Cav-1 and stress signals is warranted.

Effects of Traditional Chinese Medicine on Chemotherapy-Induced Myelosuppression and Febrile Neutropenia in Breast Cancer Patients.

Title. Chemotherapy-induced myelosuppression lowers the quality of life in breast cancer patients and causes many complications. Traditional Chinese Medicine (TCM) is a widely used complementary and alternative medicine therapies. Objective. To study whether TCM can reduce the incidence of chemotherapy-induced leukopenia, neutropenia, and febrile neutropenia (FN) in breast cancer patients. Methods. The data were analyzed retrospectively between patients who received TCM treatment (group 1, n = 453) and patients who did not receive TCM treatment (group 2, n = 359). Significant risk factors associated with the occurrence of chemotherapy-induced leukopenia, neutropenia, and FN were identified using multivariate analysis. Propensity score-matched patients were analyzed to adjust for any baseline differences. Results. Group 1 patients had a significantly lower rate of chemotherapy-induced severe leukopenia, neutropenia, and FN, compared with group 2 (43% versus 71%, P < 0.0001, 72% versus 78%, P = 0.005, 6% versus 24%, P < 0.0001, resp.). Multivariate analysis revealed that chemotherapy regimens containing anthracyclines combined with paclitaxel or docetaxel were the most significant predictor. Subgroup analysis indicated that TCM treatment showed benefit in relieving chemotherapy-induced leukopenia and FN in most chemotherapy regimens. Conclusions. TCM treatment could lower the risk of severe chemotherapy-induced leukopenia, neutropenia, and FN in breast cancer patients.

Caveolin-1 mediates chemoresistance in breast cancer stem cells via ß-catenin/ABCG2 signaling pathway.

Accumulating evidence has suggested that cancer stem cells (CSCs) are at the root of drug resistance, and recent studies have indicated that caveolin-1, a membrane transporter protein, is involved in the regulation of cancer chemoresistance and stem cell signaling. However, the current understanding of the role of caveolin-1 in breast cancer development remains controversial. Herein, we demonstrate that caveolin-1 expression was upregulated after breast cancer chemotherapy in vitro and in vivo, accompanied by co-overexpression of ß-catenin and ATP-binding cassette subfamily G member 2 (ABCG2) signaling. Additionally, breast CSCs were enriched for caveolin-1 expression. Caveolin-1 silencing sensitized breast CSCs by limiting their self-renewal ability but promoting the differentiation process. ß-catenin silencing prevented the enhanced chemoresistance of CSCs induced by caveolin-1 overexpression, indicating that ß-catenin is an essential molecule responsible for caveolin-1-mediated action. Further mechanistic investigation revealed that caveolin-1 silencing could downregulate the ß-catenin/ABCG2 pathway through glycogen synthase kinase 3 beta activation and Akt inhibition, resulting in increased ß-catenin phosphorylation and proteasomal degradation. Clinical investigation also revealed a close correlation between caveolin-1 and ß-catenin/ABCG2 signaling in breast cancer samples. Notably, caveolin-1 was highly elevated in triple-negative breast cancer, and caveolin-1 silencing significantly impaired the tumorigenicity and chemoresistance of breast CSCs in in vivo models. Overall, our study not only highlights the role of caveolin-1 in mediating the chemoresistance of breast CSCs via ß-catenin/ABCG2 regulation but also provides novel approaches for future therapies targeting CSCs.

MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin.

Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.

[Effect of Astragalus mongholicus injection on proliferation and apoptosis of hormone sensitive (MCF-7) breast cancer cell lines with physiological dose E2].

OBJECTIVE: To investigate the effect of Astragalus mongholicus injection on proliferation and apoptosis in hormone sensitive breast cancer cell lines (MCF-7) with physiological dose E2. METHODS: Cell control group, TAM control group and five different dose Astragalus mongholicus injection groups with physiological dose E2 were set in the study. Proliferation of MCF-7 cells were evaluated with MTT assay, cell apoptotic rate were measured with flow cytometry, DNA ladder and cell cycle were observed. RESULTS: In physiological dose E2, Astragalus mongholicus injection inhibited MCF-7 cells proliferation at all concentration groups. As time lasting, Astragalus mongholicus injection showed better inhibitory effect than TAM (P<0.05). Among 2 x 10(-1) g/mL-2 x 10(-4) g/mL concentration, Astragalus mongholicus injection significantly increased the proliferative percent of G0/G1 and S-phase cell, decreased percent of G2-M phase cell (P<0.05) at 24 hours. After cocultured 72 hours, Astragalus mongholicus injection increased the rate of apoptosis to 16.7% at 2 x 10(-1) g/mL concentration. CONCLUSION: In some dose limit, Astragalus mongholicus injection may inhibit proliferation, induce apoptosis and interrupt caryocinesia at G0-G1 phase or S phase in hormone sensitive (MCF-7) breast cancer cell lines with physiological dose E2.

[Standardization of syndrome differentiation based on stages for breast cancer: a significant and updating topic on mastology of traditional Chinese medicine].

The incidence of breast cancer increased rapidly in recent years. Breast cancer has become the most frequent malignant tumor of female especially in the developed regions. Traditional Chinese medicine (TCM) is effective in treating breast cancer, but its theories appear hysteretic, restricting the progress in clinical practice, teaching and research of TCM in the treatment of breast cancer. This article described the significance and urgency to work out the standardization of syndrome differentiation based on stages for breast cancer and put it into practice. It also analyzed the foundations, ideas and approaches of the research of standardization of syndrome differentiation based on stages for breast cancer in light of the changes of spectrum of diseases, the weaknesses of modern medicine in treating breast cancer, and the existed problems in the update clinical practice.

[Discussion of some problems about use of traditional Chinese medicine in perioperative management of breast cancer].

The study on use of traditional Chinese medicine in perioperative management of breast cancer is still in the beginning phase. With the emergence of new understanding about the biological characteristics of breast cancer, the concept of treatment has changed. For instance, the resection extent is tending to be narrower, large doses of radiotherapy may be adopted during the operation, and early use of adjuvant chemotherapy is advocated after the operation. These have bought about changes to the intervention factors in the perioperative period for breast cancer. Some related problems about the participation of traditional Chinese medicine in perioperative management of breast cancer are discussed in this article, so as to make the perioperative management perfect.

[Effects of Runing Recipe II on expressions of p53 and ras oncogene proteins and cell cycle of the transplanted Ca761 breast cancer in mice].

OBJECTIVE: To explore the mechanisms of Runing Recipe II (a recipe composed of traditional Chinese herbs) in inhibiting the growth of breast cancer by observing its effects on the expressions of p53 and ras oncogene proteins and cell cycle of the transplanted Ca761 breast cancer in mice. METHODS: We established the breast cancer model by transplanting Ca761 cells in mice. The mice were randomly divided into 4 groups: normal saline control group, CTX-treated group, Runing Recipe II-treated group, and Runing Recipe II and CTX-treated group, with 12 mice in each group. We detected the cell cycle of the cancer cells in the mice's transplanted tumor with flow cytometry and measured the expressions of p53 and ras oncogene proteins in the transplanted tumor with immunohistochemical methods. RESULTS: The percentages of tumor cells in S-phase of the Runing Recipe II treated group, CTX-treated group and Runing Recipe II and CTX-treated group were significantly lower than that of the normal saline control group respectively (P<0.05). The percentage of tumor cells in G(0)-G(1) phase of the Runing Recipe II treated group was lower than that of the CTX-treated group (P<0.05), while the percentage of tumor cells in G(2)-M phase was higher than that of the CTX-treated group. The immunoreactive scores (IRSs) of p53 in the Runing Recipe II treated group and Runing Recipe II and CTX-treated group were significantly lower than that in the normal saline control group respectively (P<0.05). The effect of CTX on the expression of p53 was not significant. The IRSs of ras oncogene protein in the Recipe II-treated group, CTX-treated group and Runing Recipe II and CTX-treated group were lower than that in the normal saline control group respectively (P<0.05). CONCLUSION: Runing Recipe II can inhibit the growth of Ca761 breast cancer in mice by controlling the cell cycle of the transplanted tumor. This may be related to its effect on the gene expressions of p53 and ras in the tumor tissue.

[Study progress in reversing multidrug resistance to breast cancer with Chinese herbs].

Multidrug resistance of breast cancer is one of the most causes of failure in clinical chemotherapy. It is important to find out some safe and effective drugs to reserve multidrug resistance to breast cancer. The effect of some herbs had been identified in vitro. This article mainly reviewed the research progress in reversing multidrug resistance to breast cancer with Chinese herb. If the herb's effects and safety can be testified in vivo by further research, it will be effectively applied in clinic.