Dual-Labeled Single Fluorescent Probes for the Simultaneous Two-Color Visualization of Dual Organelles and for Monitoring Cell Autophagy.

Dual-labeled single fluorescent probes are powerful tools for studying autophagy on the molecular scale, yet their development has been hampered by design complexity and a lack of valid strategies. Herein, for the first time, we introduce a combinatorial regulation strategy to fabricate dual-labeled probes for studying autophagy by integrating the specific organelle-targeting group and the functional fluorescence switch into a pentacyclic pyrylium scaffold (latent dual-target scaffold). For proof of concept, we prepared a range of dual-labeled probes (TMOs) that display different emission colors in duple organelles. In these probes, TMO1 and TMO2 enabled the simultaneous two-color visualization of the lysosomes and mitochondria. The other probes (TMO3 and TMO4) discriminatively targeted lysosomes/nucleolus and lysosomes/lipid droplets (LDs) with dual-color emission characteristics, respectively. Intriguingly, by simply connecting the endoplasmic reticulum (ER) targeting group to the pentacyclic pyrylium scaffold, we created the first dual-labeled probe TMO5 for simultaneously labeling lysosomes/ER in distinctive fluorescent colors. Subsequently, using the dual-labeled probe TMO2, drug-induced mitophagy was successfully recorded by evaluating the alterations of multiple mitophagy-related parameters, and the mitophagy defects in a cellular model of Parkinson's disease (PD) were also revealed by simultaneous dual-color/dual-organelle imaging. Further, the probe TMO4 can track the movement of lysosomes and LDs in real time and monitor the dynamic process of lipophagy. Therefore, this work not only presents attractive dual-labeled probes to promote the study of organelle interactions during autophagy but also provides a promising combinatorial regulation strategy that may be generalized for designing other dual-labeled probes with multiple organelle combinations.

Slippery Au Nanosphere Monolayers with Analyte Enrichment and SERS Enhancement Functions.

Slippery surfaces can enrich analytes from solutions into tiny dots after solvent evaporation for surface-enhanced Raman scattering (SERS) detection. Here, we make the self-assembled Au nanosphere monolayers slippery, which can not only behave as SERS substrates but also enrich the analytes during solvent evaporation. A thin silica shell was used to wrap the Au nanosphere monolayer to allow the functionalization of a slippery polydimethylsiloxane brush monolayer onto it. These slippery Au nanosphere monolayers could be easily cleaned and reused many times. When Au nanospheres were introduced into the analyte solution droplet on the slippery Au nanosphere monolayer, a 3D Au nanoparticle/analyte aggregate was formed after solvent evaporation. Both the Au nanoparticle aggregate and the underneath slippery Au nanosphere monolayer could contribute to SERS enhancement. We endow the self-assembled Au nanosphere monolayer SERS substrates with an analyte enrichment function, greatly strengthening their SERS enhancement.

PARP inhibitor treatment of advanced breast cancer beyond the BRCA-mutated type: a meta-analysis.

Background: We conducted this meta-analysis to compare the efficacy and safety of PARP inhibitors with or without chemotherapy versus chemotherapy alone for advanced breast cancer. Methods: A meta-analysis and trial sequential analysis were performed using RevMan 5.2 analysis software. Results: Six eligible randomized clinical trials involving 2080 patients were included. Regimens containing PARP inhibitors were significantly associated with higher objective response rate, longer progression-free survival and overall survival. The PARP inhibitor regimen group had a significantly higher rate of grade ≥3 thrombocytopenia than the chemotherapy-only group. Conclusion: Regimens containing PARP inhibitors are effective and safe for BRCA-mutated advanced breast cancer patients. The efficacy appears to be only marginal in patients with BRCA status unselected.

Lay abstract The present meta-analysis was conducted to compare the efficacy and safety of PARP inhibitors with or without chemotherapy versus chemotherapy alone for advanced breast cancer. Six eligible randomized clinical trials involving 2080 patients were included. Regimens containing PARP inhibitors were significantly associated with higher objective response rate, longer progression-free survival and better overall survival, but a higher rate of grade ≥3 thrombocytopenia. Regimens containing PARP inhibitors are effective and safe for patients with advanced breast cancer who have a BRCA gene mutation. The efficacy appears to be only marginal in patients with BRCA status unselected.

Distinctive features of immunostaining and mutational load in primary pulmonary enteric adenocarcinoma: implications for differential diagnosis and immunotherapy.

BACKGROUND: Primary pulmonary enteric adenocarcinoma (PEAC) is an extremely rare variant of invasive lung cancer. It is highly heterogeneous while shares some common morphologic and immunohistochemical features with usual pulmonary adenocarcinoma (PAC) and colorectal adenocarcinoma (CRAC), making the differential diagnosis difficult. At present there are only limited studies about distinctive features of primary PEAC and the results are often inconsistent. METHODS: We retrospectively analyzed total 129 primary PEACs and 50 CRACs that were published since 1991 or diagnosed in our centre. Among them eight typical samples of primary PEACs and usual PACs were detected by targeted exome sequencing. RESULTS: The combination of CK7+/CDX2+ acquires high sensitivity (71.3%) and specificity (82%) in differential diagnosis of PEACs from CRAC. The primary PEACs harbor a high incidence of KRAS mutation but almost absent of EGFR mutation. Moreover, compared with usual PACs, the primary PEACs have higher nonsynonymous tumor mutation burden and more frequent MMR mutation. CONCLUSIONS: The combination of CK7+/CDX2+ immunostaining and the distinctive genetic signatures, including low incidence of sensitivity genes mutations and high tumor mutation burden, is an important supplementary to the clinical differential diagnosis of primary PEACs. Our findings thus have significant implications for development of individualized treatment strategy in these patients.

[Advances on correlation of PET-CT findings with breast cancer molecular subtypes, treatment response and prognosis].

In recent years, PET-CT has an increasing importance in the diagnosis and treatment of breast cancer. PET-CT scan can be used as a noninvasive method for molecular subtyping of breast cancer, and prediction of therapeutic effect and prognosis of patients. Studies have revealed that luminal A subtype has a significantly lower maximum standard intake value (SUVmax) than the other subtypes; triple-negative and human epidermal growth factor receptor 2 (HER2) positive tumors have relatively high SUVmax than luminal B subtype, but the specificity and sensitivity of SUVmax in diagnosis of molecular subtypes are very low, so its clinical application is limited. In predicting the effectiveness of the treatment and the prognosis of the patients, the decreased uptake of fluorodeoxyglucose (FDG) is correlated with better therapeutic effect. In addition, patients with high FDG uptake have worse survival outcomes. New tracers, such as 18F-fluoroestradiol (18F-FES) and[89Zr]trastuzumab play an important role in molecular subtyping of breast cancer. 18F-FES PET-CT can effectively evaluate the estrogen receptor (ER) status of breast cancer and the response to endocrine therapy.[89Zr]trastuzumab PET-CT can evaluate the expression of HER2 and localization of HER2-overexpressing tumors, but their specificities and sensitivities are also low. In this article, we review the recent advances on the correlation of PET-CT findings with molecular subtypes, treatment response and prognosis of breast cancer.

Increased circulating Lin(-/low) CD33(+) HLA-DR(-) myeloid-derived suppressor cells in hepatocellular carcinoma patients.

AIM: Myeloid-derived suppressor cells (MDSC) can be induced or expanded in tumor-bearing mice and cancer patients. The frequency of MDSC denoted here as Lin(-/low) CD33(+) HLA-DR(-) was investigated in hepatocellular carcinoma (HCC) patients. The clinical relevance of MDSC and patients' characteristics were examined. Also, MDSC-related immune regulatory pathways in these patients were discussed. METHODS: The quantity of MDSC was tested in peripheral blood of patients with HCC (n = 63) and healthy donors (n = 56). The expressions of interferon (IFN)-γ, vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-13, nitric oxide synthase (NOS)-2 and arginase (ARG)-1 were analyzed. Co-culturing with anti-CD3/CD28-stimulated T lymphocytes was used to determine the suppressive effect of MDSC on the T lymphocytes. RESULTS: Patients with treatment-naive HCC had an increased subpopulation of Lin(-/low) CD33(+) HLA-DR(-) cells in the peripheral blood mononuclear cells (PBMC) with characteristics of MDSC and associated to the stage (P = 0.0004). Patients with splenomegaly had a higher frequency of circulating MDSC. Also, COX-2, MMP-13 and VEGF were expressed differently associated with the alteration of MDSC. CONCLUSION: Our study provides evidence showing an increased population of Lin(-/low) CD33(+) HLA-DR(-) MDSC in the peripheral blood of HCC patients. Our data also suggest that MMP-13 and COX-2 in PBMC may play a new important role companied with MDSC in HCC patients.

Fatty Acid Metabolism: A New Perspective in Breast Cancer Precision Therapy.

Breast cancer has a special tumor microenvironment compared to other solid tumors, which is usually surrounded by a large number of adipocytes that can produce and secrete fatty acids and adipokines. Adipocytes have a remodeling effect on breast cancer lipid metabolism, while fatty acids and lipid droplets can make breast cancer cells more aggressive. Lipid metabolism, especially the synthesis of fatty acids, is an important cellular process for membrane biosynthesis, energy storage, and signal molecule production. Therefore, blocking the lipid supply to cancer cells or changing the lipid composition has an important impact on the signal transmission and cell proliferation of cancer cells. Alterations in lipid availability can also affect cancer cell migration, induction of angiogenesis, metabolic symbiosis, evasion of immune surveillance, and cancer drug resistance. Fatty acid synthesis and metabolism have received extensive attention as potential targets for cancer therapy, and studies on modulating the tumor lipid microenvironment to improve the sensitivity of antitumor drugs have also been discussed; however, strategies to target these processes have not been translated into clinical practice.

Clinical and molecular profiling of EGFR-mutant lung adenocarcinomas transformation to small cell lung cancer during TKI treatment.

Introduction: Small cell lung cancer (SCLC) transformation serves as a significant mechanism of resistance to tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. To address this clinical challenge, we conducted a retrospective analysis at Zhejiang University School of Medicine, the First Affiliated Hospital, focusing on patients with EGFR sensitizing mutations. Methods: A total of 1012 cases were included in this retrospective analysis. The cohort primarily consisted of patients with EGFR sensitizing mutations. Biopsy-confirmed small cell transformation was observed in seven patients, accounting for 0.7% of the cases. All patients in this subset were initially diagnosed with stage IV adenocarcinoma (ADC), with four cases classified as poorly differentiated and three as moderately to poorly differentiated ADC. EGFR exon 19 deletions were identified in five of these cases. Next-generation sequencing (NGS) was performed on seven cases, revealing mutations in the tumor protein p53 (TP53) gene in four cases and loss of the retinoblastoma1 (RB1) gene in three cases. Results: The median duration from the initial diagnosis to small cell transformation was 35.9 months (interquartile range: 12.1-84 months). Following small cell transformation during EGFR inhibition, all patients received etoposide/platinum-based treatment, leading to a median progression-free survival (PFS) of 4.7 months (interquartile range: 2.7-10.1 months). Notably, most patients in this series had poorly differentiated adenocarcinomas at the outset. TP53 mutations and RB1 loss were common genetic alterations observed in patients with small cell transformation in this cohort. Discussion: The findings underscore the clinical significance of SCLC transformation as a resistance mechanism to EGFR TKIs in NSCLC with EGFR mutations. The observed genetic alterations, including TP53 mutations and RB1 loss, suggest potential associations with the transformation process and warrant further investigation. Understanding the genetic landscape and clinical outcomes in patients experiencing small cell transformation can contribute to improved strategies for managing resistance in EGFR-mutant NSCLC.

HER2 changes to positive after neoadjuvant chemotherapy in breast cancer: A case report and literature review.

BACKGROUND: As the most common cancer in women, breast cancer is the leading cause of death. Most patients are initially diagnosed as stage I-III. Among those without distant metastases, 64% are local tumors and 27% are regional tumors. Patients in stage IIA-IIIC and those who meet the breast-conserving criterion with the exception of tumor size can consider neoadjuvant chemotherapy (NACT). It is worth noting that the status of tumor cell biomarkers is not consistently static. Endocrine-related estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) encoded by erythroblastic leukemia viral oncogene homolog 2 gene can all alter from positive to negative or vice versa, especially in luminal B subtype after NACT. In addition, determination of HER2 status currently mainly relies on immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), but FISH is commonly used when the result of IHC is uncertain. HER2 is regarded as negative when the IHC result is 0/1+ without the addition of FISH. To the best of our knowledge, this is the first report of a case harboring HER2 status transformation and IHC1+ with positive amplification by FISH after NACT. CASE SUMMARY: A 49-year-old woman discovered a mass in her right breast and underwent diagnostic workup. Biopsies of the right breast lesion and axillary lymph nodes were obtained. The results pointed to invasive ductal carcinoma with the IHC result for ER (80%), PR (60%), Ki-67 (20%) and ambiguous expression of HER2 (IHC 2+) with negative amplification by FISH (HER2/CEP17 ratio of 1.13). She underwent surgery after NACT. The pathological findings of the surgically resected sample supported invasive ductal carcinoma with the tumor measuring 1.1 cm × 0.8 cm × 0.5 cm and had spread to one of fifteen dissected lymph nodes. Retesting of the specimen showed that the tumor was positive for ER (2+, 85%) and PR (2+, 10%) but negative for HER2 by IHC (1+). Also Ki-67 had dropped to 2%. The patient was regularly monitored every 3 mo without evidence of recurrence. CONCLUSION: Biomarker status should be reassessed after NACT especially in luminal subtypes.

Long survival after immunotherapy plus paclitaxel in advanced intrahepatic cholangiocarcinoma: A case report and review of literature.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary hepatic malignancy worldwide. However, currently available systemic therapies are of limited effectiveness, and the median overall survival of patients treated with first-line standard chemotherapy is less than one year. Immune checkpoint inhibitors have been used to treat solid tumors. Clinical studies recently explored the combination of chemotherapy and immunotherapy for CCA. However, the clinical significance of predictive biomarkers for chemo-immunotherapy in CCA remains unclear. It is also worth exploring whether a combination of chemotherapeutic agents can increase the sensitivity of CCA immunotherapy. CASE SUMMARY: This study reports a case of advanced iCCA in which clinical complete remission had been achieved using a programmed death 1 (PD-1) inhibitor and paclitaxel without known predictive biomarkers, but with BRCA1, KRAS, and NTRK3 mutations after rapid progression to first-line chemotherapy, and has remained in clinical complete remission for more than two years. This case suggests that chemo-immunotherapy is a potential therapeutic option for patients with iCCA and few known predictive biomarkers for immunotherapies as well as synergistic effect of the combination of paclitaxel and PD-1 monoclonal antibody. CONCLUSION: The combination of paclitaxel and PD-1 monoclonal antibodyr can be explored in patients with advanced iCCA.

Alpha-fetoprotein-producing hepatoid adenocarcinoma of the lung responsive to sorafenib after multiline treatment: A case report.

BACKGROUND: Hepatoid adenocarcinoma of the lung (HAL) is an extremely rare malignant tumor, and many patients with HAL exhibit high levels of alpha-fetoprotein (AFP) expression. Currently, there is no standardized treatment strategy for advanced HAL and its prognosis is poor. CASE SUMMARY: We report a 55-year-old man with unresectable AFP-related HAL. The largest cross-sectional area of the mass in the upper lobe of the left lung at the beginning of treatment was 8.46 cm × 6.53 cm. The patient's serum AFP level was 9283 ng/mL. The mass increased in size to 8.86 cm × 8.21 cm after two courses of platinum-based combination chemotherapy and immunotherapy, and serum AFP reached its highest level (71232.2 ng/mL). The patient was treated with sorafenib (400 mg twice daily, per os). Forty days later, the mass was reduced to 5.63 cm × 5.29 cm and serum AFP level dropped to 786.8 ng/mL. The patient achieved partial remission for > 9 mo with sorafenib and an excellent biomarker response, as well as survival > 13 mo, which is among the longest reported for unresectable stage IV HAL. CONCLUSION: This is the first report to document successful treatment of unresectable AFP-related HAL with single-agent sorafenib after multiline therapy.

Rational engineering of biomimetic flavylium fluorophores for regulating the lysosomal and mitochondrial localization behavior by pH-induced structure switch and application to fluorescence imaging.

Mitochondria and lysosomes, as the important subcellular organelles, play vital roles in cell metabolism and physiopathology. However, there is still no general method to precisely regulate the lysosomal and mitochondrial localization behavior of fluorescent probes except by selecting specific targeting groups. Herein, we proposed a pH-induced structure switch (pHISS) strategy to solve this tricky puzzle. For the proof-of-concept, we have rationally designed and synthesized a series of cationic flavylium derivatives FL-1-9 with tunable pH-induced structure switch through adjusting the electron-donating ability of the substituents. As expected, the co-localization imaging experiments revealed that the lysosomal and mitochondrial localization behavior of FL-1-9 dyes is closely related to their pHISS ability. It is noteworthy that FL cationic dyes with strong electron-donors are not prone to pHISS and can be well enriched in mitochondria, while FL cationic dyes with weak electron-donors are highly susceptible to pHISS and display an unusual lysosome-targeting capability. This also provided a feasible strategy for lysosomal localization without basic groups and presented new application options for some flavylium dyes previously thought to be less stable. Furthermore, FL cationic dyes with medium electron-donor exhibit certain localization abilities both in mitochondria and lysosomes. Finally, through a detailed study of pH-induced structure switch and exploiting the pH inertia brought by the strong electron-donors, a novel NIR ratiometric fluorescent probe with large wavelength-shift was constructed for monitoring mitochondrial H2S in living cells, tumor tissues and living mice, highlighting the value of the pHISS strategy in precisely regulating organelle targeting and constructing corresponding organelle targeting probes.

Sporoderm-Broken Spores of Ganoderma lucidum Sensitizes Ovarian Cancer to Cisplatin by ROS/ERK Signaling and Attenuates Chemotherapy-Related Toxicity.

Although platinum-based chemotherapeutics such as cisplatin are the cornerstone of treatment for ovarian cancer, their clinical application is profoundly limited due to chemoresistance and severe adverse effects. Sporoderm-broken spores of Ganoderma lucidum (SBSGL) have been reported to possess antitumor effects. However, the function and mechanism of SBSGL and its essential composition, ganoderic acid D (GAD), in the cisplatin therapy on ovarian cancer have yet to be investigated. Here, we investigated the combined effect of SBSGL and cisplatin in an ovarian tumor xenograft model. The results showed that combining SBSGL with cisplatin reduced tumor growth and ameliorated cisplatin-induced intestinal injury and myelosuppression. We also confirmed that GAD could enhance the therapeutic effect of cisplatin in SKOV3 and cisplatin-resistant SKOV3/DDP cells by increasing the intracellular reactive oxygen species (ROS). Mechanistically, we proved that ROS-mediated ERK signaling inhibition played an important role in the chemo-sensitization effect of GAD on cisplatin in ovarian cancer. Taken together, combining SBSGL with cisplatin provides a novel therapeutic strategy against ovarian cancer.

PITX2 methylation: a novel and effective biomarker for monitoring biochemical recurrence risk of prostate cancer.

AIMS: Prostate cancer is one of the most common malignancies in men. Biochemical recurrence (BCR) and progression following curative treatment pose a significant public health challenge. Thus, it is essential to explore effective biomarkers for disease progression monitoring and risk stratification. The promoter region of the paired-like homeodomain transcription factor 2 (PITX2) gene has been found to be frequently methylated in prostate cancer. However, the prognostic role of PITX2 methylation in prostate cancer and which patients most likely to be recommended for PITX2 methylation tests to assess BCR risk remain controversial. Therefore, a systematic review was performed to explore the relationship of PITX2 methylation with the BCR risk of prostate cancer. METHODS: The PubMed, EMBASE, and Cochrane Library databases were systematically searched for eligible studies. Seven studies with a total of 2185 patients were included. Pooled hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were calculated. RESULTS: The overall HR was 2.71 (95% CI, 2.21-3.31), suggesting that PITX2 methylation has an adverse impact on BCR of prostate cancer. The pooled estimate of 5-year BCR-free survival for patients with a high methylation status was significantly lower than that for patients with a low methylation status (71% vs 90%; odds ratio [OR] = 3.50; 95% CI, 2.67-4.60, P = .000). A subgroup analysis was conducted according to detection method; the combined HRs were 2.68 (95% CI, 2.02-3.55) for quantitative methylation-specific PCR (qMSP) and 3.29 (95% CI, 2.31-4.68) for microarray EpiChip. In subgroups defined by region, Gleason score, pathological stage, surgical margin status and ethnicity, high methylation status was also associated with BCR of prostate cancer. CONCLUSIONS: As an effective biomarker, PITX2 methylation is feasible for individualized BCR risk assessment of prostate cancer following radical prostatectomy.

Anti-PD-1/PD-L1 antibodies versus docetaxel in patients with previously treated non-small-cell lung cancer.

Anti-PD-1/PD-L1 antibodies have been proved one of the most promising treatments against non-small cell lung cancer (NSCLC); however, whether anti-PD-1/PD-L1 antibodies can provide added benefits for pretreated patients with advanced NSCLC and which patients are most likely to benefit from anti-PD-1/PD-L1 therapy remain controversial. This meta-analysis evaluated the efficacy and safety between anti-PD-1/PD-L1 antibodies and docetaxel in previously treated, advanced NSCLC. PubMed, EMBASE and Cochrane library databases were systematically searched for eligible studies. Five studies with a total of 3,025 patients were included. Our results showed that, for all patients, anti-PD-1/PD-L1 therapy prolonged overall survival (OS) (hazard ratio [HR] = 0.69; 95% CI, 0.63-0.75) and progression-free survival (PFS) (HR = 0.87; 95% CI, 0.80-0.94). For patients with PD-L1 expression ≥1%, anti-PD-1/PD-L1 therapy had higher objective response rates. In subgroup analysis according to the tumor PD-L1 expression level, anti-PD-1/PD-L1 therapy was associated with longer OS and PFS in patients with high PD-L1 expression (≥1%, ≥5%, ≥10% and ≥50%), but not in those with low expressions. In subgroup analysis of patients' characteristics, anti-PD-1/PD-L1 antibodies showed OS benefits across most prespecified subgroups, except for patients with EGFR mutation-positive and never smokers. For patients with EGFR mutation, anti-PD-1/PD-L1 therapy was an unfavorable factor of PFS. The grade 3 or 4 adverse events rates of anti-PD-1/PD-L1 treatment were significantly lower than that of docetaxel. Our results suggest that anti-PD-1/PD-L1 therapy significantly improves survival compared with docetaxel in patients with previously treated, PD-L1-positive, advanced NSCLC, and has a distinct safety profile from chemotherapy.

Inhibition of ERα/ERK/P62 cascades induces "autophagic switch" in the estrogen receptor-positive breast cancer cells exposed to gemcitabine.

Several clinical trials revealed that estrogen receptor (ER) status had relevance to the response of mammary malignancy to chemotherapy. Autophagy has emerged as an important cellular mechanism of tumor cells in response to anticancer therapy. The aim of this study is to investigate whether gemcitabine induces autophagy, and more importantly, whether such autophagy is functional relevant to the therapeutic effects of gemcitabine in breast cancer cells in relation to the ER status. In our study, autophagy was induced both in ER+ MCF-7 and ER- MDA-MB-231 cells by gemcitabine markedly, while the autophagy plays distinct roles - cytoprotective in ER- MDA-MB-231 and cytotoxic in ER+ MCF-7 cells. Gemcitabine treatment leads to the activation of ERα-ERK-P62 signal pathway in MCF-7 cells which may augment the autophagic degradation, thus results in the excessive activation of autophagy and irreversible autophagic cell death eventually. Inhibition of ERα-ERK-P62 cascades in MCF-7 cells by small interfering RNA or PD98059 impairs the autophagic degradation, and leads to "autophagic switch" - from cytotoxic autophagy to cytoprotection. Moreover, stable overexpression of ERα in the ER- BCap37 breast cancer cell line enhances the gemcitabine-induced autophagy flux and switches the autophagic cytoprotection in ER- BCap37 to cytotoxicity effect in ER+ BCap37 cells. Our study firstly demonstrated that ER status influences gemcitabine efficacy via modulating the autophagy in breast cancer cells.

The cytoprotective role of gemcitabine-induced autophagy associated with apoptosis inhibition in triple-negative MDA-MB-231 breast cancer cells.

Triple-negative breast cancer (TNBC), which is estrogen receptor (ER)-negative, progesterone receptor­negative and is also negative for HER2 expression, remains a great clinical challenge due to its strong resistance to chemotherapy at the late stage of treatment and relatively unfavorable prognosis. Gemcitabine has been approved by the FDA/SFDA for use as a first-line therapeutic drug against advanced or metastatic breast cancer. Therefore, the clarification of the mechanisms underlying gemcitabine-acquired resistance is of particular importance for the optimal management of TNBC. A number of studies have revealed that autophagy, which has been found to protect cancer cells from anti-cancer drug-induced death, may contribute to the development of drug resistance. However, the association between autophagy and gemcitabine treatment in TNBC cells has yet to be defined. Our study clearly demonstrates that gemcitabine is able to induce mTOR-independent autophagy in human triple­negative MDA-MB-231 breast cancer cells. In addition, we demonstrate that autophagy protects MDA-MB-231 cells from gemcitabine-induced cell growth inhibition and apoptosis, indicating that gemcitabine can activate autophagy to impair the sensitivity of MDA-MB­231 cells. Furthermore, as shown by our results, the inhibition of gemcitabine-induced autophagy by chloroquine shifts the expression of the p53 protein, Bcl-2 family proteins and the relative Bax/Bcl-xL ratio in favor of promoting apoptosis. These results reveal that the inhibition of apoptosis may be one of the mechanisms of autophagy-induced cytoprotection in gemcitabine-treated MDA-MB-231 cells. The apoptotic and autophagic processes constitute a mutual inhibition system and jointly seal the fate of TNBC cells that are exposed to gemcitabine. Thus, our study suggests that the combination of an autophagic inhibitor and gemcitabine as a therapeutic strategy may represent a promising approach with greater clinical efficacy for patients with TNBC. However, extended preclinical trials are required to further determine the positive effects of the inhibition of autophagy on the efficacy of gemcitabine.