Shengrong Sun

An autophagy inhibitor enhances the inhibition of cell proliferation induced by a proteasome inhibitor in MCF-7 cells

The ubiquitin-proteasome system and the autophagy-lysosome pathway are the two main routes for eukaryotic intracellular protein clearance. Inhibition of proteasome activity leads to cell death. Due to the dual roles of autophagy in tumor cell survival and death, the effect of suppression of autophagy on breast cancer cells remains to be elucidated. We investigated whether inhibition of the proteasome is capable of inducing autophagy, and we assessed the effect of combined inhibition of the proteasome and autophagy on human breast cancer MCF-7 cells. The proteasome inhibitor bortezomib was used to induce autophagy in MCF-7 cells, and the effect of autophagy on the proliferation of MCF-7 cells was investigated using the autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. The expression of autophagy‑related proteins was determined by Western blot analysis and the GFP-LC3 redistribution was detected using a fluorescence microscope after MCF-7 cells were infected with a GFP-LC3-expressing adenovirus. MCF-7 cell proliferation was inhibited and autophagy was activated in the same dose‑dependent manner. Bortezomib induced a dose‑dependent increase in LC3-II. However, when MCF-7 cells were co-treated with bortezomib and 3-MA, 3-MA blocked the increase in LC3-II protein expression and led to a significant inhibition of cell proliferation. Inhibition of the proteasome may induce autophagy in human breast cancer MCF-7 cells and 3-MA could inhibit autophagy induced by the proteasome inhibitor. A combination of 3-MA and bortezomib increases cell death. These findings indicate that suppression of the two intracellular degradation systems may shed new light on breast cancer control.

PKCδ-dependent Activation of the Ubiquitin Proteasome System is Responsible for High Glucose-induced Human Breast Cancer MCF-7 Cell Proliferation, Migration and Invasion

Type 2 diabetes mellitus (T2DM) has contributed to advanced breast cancer development over the past decades. However, the mechanism underlying this contribution is poorly understood. In this study, we determined that high glucose enhanced proteasome activity was accompanied by enhanced proliferation, migration and invasion, as well as suppressed apoptosis, in human breast cancer MCF-7 cells. Proteasome inhibitor bortezomib (BZM) pretreatment mitigated high glucose-induced MCF-7 cell growth and invasion. Furthermore, high glucose increased protein kinase C delta (PKC?)-phosphorylation. Administration of the specific PKC? inhibitor rottlerin attenuated high glucose-stimulated cancer cell growth and invasion. In addition, PKC? inhibition by both rottlerin and PKC? shRNA significantly suppressed high glucose-induced proteasome activity. Our results suggest that PKC?-dependent ubiquitin proteasome system activation plays an important role in high glucose- induced breast cancer cell growth and metastasis.

Role for ezrin in breast cancer cell chemotaxis to CCL5.

This study was undertaken to observe the effects and the possible mechanism of membrane-cytoskeleton linker ezrin on the chemokine CC chemokine ligand 5 (CCL5)-induced invasive ability in human breast carcinoma MCF-7 cells. RNA interference (RNAi) using ezrin small hairpin RNAs (ezrin shRNA) was used to analyze the role of ezrin in the regulation of this CCL5-induced malignant behavior of MCF-7 cells. The effects of recombinant human CCL5 (rhCCL5) on the cells invasive ability were detected by transwell assay. Western blotting was performed to examine the expression of the total and the phosphorylated ezrin at protein level. The CCL5-induced changes in the organization of the actin cytoskeleton in the transfected cells were determined using confocal microscopy. Flow cytometry was used to detect the cell cycle. MTT method was used to detect the proliferation of the cells. We found that the MCF-7 cells responded chemotactically to CCL5 in a dose- and time-dependent manner. After RNAi treatment, the proliferation was inhibited and the cell proportion in G2-M phase decreased. The CCL5-induced cell motility and invasiveness were obviously inhibited by the silencing of ezrin. In addition, the CCL5 induced a significant up-regulation in the total and the phosphorylated ezrin expression in MCF-7 cells, whereas in the presence of ezrin silencing, the CCL5 induced only a slight increase in the total and the phosphorylated ezrin expression. CCL5 was shown to induce changes in the organization of the actin cytoskeleton and the level of F-actin in MCF-7 cells, and the silencing of ezrin could inhibit these changes. Collectively, our data further show that CCL5 induced invasiveness in MCF-7 cells. These data indicate a potential role for ezrin in the processes of the CCL5-induced breast cancer cell migration, invasiveness and metastasis. It is suggested that ezrin may act as downstream effector of CCL5 and a new anti-invasive therapeutic target for human breast cancer.

Differential phosphofructokinase‑1 isoenzyme patterns associated with glycolytic efficiency in human breast cancer and paracancer tissues

Cancers are characterized by an increasing glycolytic activity, which is called the Warburg effect. Although this phenomenon is well known, the mechanism of the enhanced rate of glycolysis in cancer has not yet been clearly recognized. The present study investigated the glycolytic rate, regulatory enzymatic activities and the expression of phosphofructokinase-1 (PFK-1) in human breast cancer and paracancer tissues. Human breast cancer tissues have an increased degree of glycolytic efficiency and regulatory enzymatic activities, which have been shown in previous studies. However, the present study identified a number of novel observations. The total PFK-1 levels were higher in human breast cancer tissues than in paracancer tissues, and further investigations revealed differential PFK-1 isoenzyme expression patterns between human breast cancer and paracancer tissues. The human breast cancer and paracancer tissues mainly expressed PFK-P and PFK-L isoforms, respectively. Linear-regression analysis showed that, depending on the pathological stage of breast cancer, the expression of PFK-P was significantly positively correlated with the activity of PFK-1. Thus, during the development of human breast cancer, the enhancement of glycolytic activity depends primarily on the conversion of the PFK-1, from PFK-L to PFK-P.

Tunicamycin-induced ER stress regulates chemokine CCL5 expression and secretion via STAT3 followed by decreased transmigration of MCF-7 breast cancer cells

Chemokine C-C motif ligand 5 (CCL5) is an important marker related to the progression of breast cancer and is upregulated in cancer cells. However, the mechanism of the overexpression of CCL5 in tumours has not yet been clarified. The present study aimed to investigate the role of endoplasmic reticulum (ER) stress in regulating CCL5 expression and its relationship with signal transducer and activator of transcription 3 (STAT3). Meanwhile, the effect of tunicamycin, a classical ER stress inducer, and CCL5 on the transmigration of human breast cancer MCF-7 cells was observed and analysed. Compared with the normal breast epithelial tissues, expression levels of CCL5, STAT3 and CHOP, an indicator of ER stress, were significantly upregulated in breast cancer tissues. In human breast cancer MCF-7 cells, ER stress activator tunicamycin increased the expression of CCL5, STAT3 and CHOP in a time- and concentration-dependent manner. Moreover, tunicamycin-induced CCL5 expression was positively related to upregulation of unphosphorylated STAT3 (U-STAT3) but negatively related to STAT3 phosphorylation at the Tyr705 site. Furthermore, ER stress inhibited CCL5 secretion and transmigration of MCF-7 cells. This study also showed that extracellular rhCCL5 induced transmigration of MCF-7 cells which was partially blocked by the CCR5 monoclonal antibody, while knockdown of endogenous expression of CCL5 did not affect the transmigration of the cells. In conclusion, ER stress induced endogenous expression of CCL5 via elevating U-STAT3 expression; however, ER stress inhibited CCL5 secretion, which in turn, decreased the transmigration of breast cancer MCF-7 cells.

Lymph node ratio is more predictive than traditional lymph node stratification in lymph node positive invasive breast cancer.

OBJECTIVE To evaluate the relationships between lymph node ratio (LNR, the ratio of positive lymph nodes in excised axillary lymph nodes) and disease-free survival (DFS) by comparing with traditional absolute positive lymph node number (pN classification) for prediction of breast cancer (BC) progrnosis. METHODS AND PATIENTS We retrospectively reviewed patients who received comprehensive therapy in Department of Breast Surgery, Hubei Cancer Hospital, China from Jan 2002 to Dec 2006 (Group A), and Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, China from Jun 2008 to May 2012 (Group B). Patients were allocated to low-risk (≤0.20), intermediate-risk (> 0.20 but ≤ 0.65), high-risk (>0.65) groups by LNR. The primary endpoint was 5-DFS. RESULTS A total of 294 patients were included in our study. LNR was verified as a negative prognostic factor for DFS (P= 0.002 in Group A, P< 0.0001 in Group B). Then we found the effects of pN and LNR delamination on disease-free survival (DFS) had statistical significance (P=0.012 for pN and P=0.031 for LNR stratification in Group A, both of them P<0.001 in Group B). Compared to pN staging, LNR staging displayed superior performance in prognosis, the adjusted hazard ratio of recurrence being 2.07 (95%CI, 1.07 to 4.0) for intermediate risk group (P=0.030) and 2.44 (95%CI, 1.21 to 4.92) for high risk group (P=0.013) in Group A. CONCLUSIONS LNR stratification proved an adverse prognostic factor of DFS in lymph nodes positive invasive BC using cut-off values 0.20 and 0.65, and was more predictive than traditional pN classification for 5-DFS.

Sonographically Guided Vacuum-Assisted Breast Biopsy for Complete Excision of Presumed Benign Breast Lesions

Benign breast disease has a high prevalence and a substantial impact on womens quality of life. Excisional biopsy is still frequently used for the diagnosis of breast masses in China. We report our experience with percutaneous removal of presumed benign lesions under sonographic guidance with a multidirectional handheld vacuum‐assisted biopsy device and evaluate this procedure as an alternative approach in the diagnosis and treatment of clinically benign breast lesions.

Clinical and sonographic assessment of cervical lymph node metastasis in papillary thyroid carcinoma.

The purpose of this study was to assess the differences in clinical and sonographic features of papillary thyroid carcinoma (PTC) between cervical lymph node metastatic (CLNM) and nonmetastatic groups. Clinical data of PTC patients (414 patients with 624 malignant nodules) who underwent a preoperative ultrasonography and surgery between June 2010 and March 2015 at Renmin Hospital of Wuhan University were retrospectively analyzed. Clinical factors, preoperative ultrasound features and the final pathological findings were obtained. The differences in the sonographic features of PTC between the CLNM group and the non-CLNM group were analyzed. There were 187 CLNM and 227 non-CLNM patients. The median age at the diagnosis of this cohort was 45.4 years old (ranging from 18 to 77 years). Ultrasonographic parameters that were significantly associated with CLNM [OR=2.569 (1.502, 4.393), P<0.001)] were as follows: the mulifocality of the nodules, size over 2 cm, the presence of microcalcifications, the distance ratio (DR) pattern showing the contact of the nodules with the thyroid capsule, and the extracapsular spread of the nodules. No significant differences in age, gender, thyroid stimulating hormone (TSH) levels and other ultrasonography parameters were found between the CLNM and the non-CLNM groups. Therefore, our results suggest that a larger size, microcalcifications, mulifocality, and the DR pattern showing the contact of the nodules with the thyroid capsule and extracapsular spread are significantly more indicative of CLNM in PTC.The purpose of this study was to assess the differences in clinical and sonographic features of papillary thyroid carcinoma (PTC) between cervical lymph node metastatic (CLNM) and nonmetastatic groups. Clinical data of PTC patients (414 patients with 624 malignant nodules) who underwent a preoperative ultrasonography and surgery between June 2010 and March 2015 at Renmin Hospital of Wuhan University were retrospectively analyzed. Clinical factors, preoperative ultrasound features and the final pathological findings were obtained. The differences in the sonographic features of PTC between the CLNM group and the non-CLNM group were analyzed. There were 187 CLNM and 227 non-CLNM patients. The median age at the diagnosis of this cohort was 45.4 years old (ranging from 18 to 77 years). Ultrasonographic parameters that were significantly associated with CLNM [OR=2.569 (1.502, 4.393), P<0.001)] were as follows: the mulifocality of the nodules, size over 2 cm, the presence of microcalcifications, the distance ratio (DR) pattern showing the contact of the nodules with the thyroid capsule, and the extracapsular spread of the nodules. No significant differences in age, gender, thyroid stimulating hormone (TSH) levels and other ultrasonography parameters were found between the CLNM and the non-CLNM groups. Therefore, our results suggest that a larger size, microcalcifications, mulifocality, and the DR pattern showing the contact of the nodules with the thyroid capsule and extracapsular spread are significantly more indicative of CLNM in PTC.