Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.     

TQFL12, a novel synthetic derivative of TQ,inhibits triple-negative breast cancer metastasis and invasion through activating AMPK/ACC pathway

Thymoquinone (TQ) has been reported as an anti-tumour drug widely studied in various tumours, and its mechanism and effect of which has become a focus of current research. However, previous studies from our laboratory and other groups found that TQ showed weak anti-tumour effects in many cancer cell lines and animal models. Therefore, it is necessary to modify and optimize the structure of TQ to obtain new chemical entities with high efficiency and low toxicity as candidates for development of new drugs in treating cancer. Therefore, we designed and synthesized several TQ derivatives. Systematic analysis, including in vitro and in vivo, was conducted on a panel of triple-negative breast cancer (TNBC) cells and mouse model to demonstrate whether TQFL12, a new TQ derivative, is more efficient than TQ. We found that the anti-proliferative effect of TQFL12 against TNBC cells is significantly stronger than TQ. We also demonstrated TQFL12 affects different aspects in breast cancer development including cell proliferation, migration, invasion and apoptosis. Moreover, TQFL12 inhibited tumour growth and metastasis in cancer cell–derived xenograft mouse model, with less toxicity compared with TQ. Finally, mechanism research indicated that TQFL12 increased AMPK/ACC activity by stabilizing AMPKα, while molecular docking supported the direct interaction between TQFL12 and AMPKα. Taken together, our findings suggest that TQFL12, as a novel chemical entity, possesses a better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.     

Dual blockade of VEGFR1 and VEGFR2 by a novel peptide abrogates VEGF-driven angiogenesis,tumor growth,and metastasis through PI3K/AKT and MAPK/ERK1/2 pathway

Background

Neutralization of vascular endothelial growth factor receptor 1 (VEGFR1) and/or VEGFR2 is a widely used means of inhibiting tumor angiogenesis.

Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway

Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24h, the IC(50) values were 27.26, 37.51 and 76.10μM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2h of MCF-7 cells with the antioxidant compound N-acetyl-l-cysteine (NAC, 5mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway.     

KLF6 inhibits estrogen receptor-mediated cell growth in breast cancer via a c-Src-mediated pathway

Estrogen receptors play a key role in breast cancer development and progression. Kruppel-like factor 6 (KLF6) is a tumour-suppressing protein. The aim of this study was to identify the role of KLF6 inhibition in estrogen receptor{alpha} (ERα)-elicited breast cancer development. Protein expression levels were examined by western blot analysis and immunoprecipitation was used to analyse interactions between proteins. An MTT assay was used to study cell proliferation. We found that KLF6 mediates cell growth in ERα-positive breast cancer cells through interaction with the c-Src protein. This interaction causes inactivation of the Erk and Akt proteins. These pathways are critical for the proliferation and survival of breast cancer cells. We also established that KLF6 could not mediate cell growth in ERα-negative cells. We conclude that KLF6 can modulate ERα-mediated cell growth in breast cancer cells. The unique role of KLF6 in mediating cell growth in breast cancer cells opens up the possibility of a new therapeutic strategy for treating breast cancer.     

Ursolic acid inhibits tumor angiogenesis and induces apoptosis through mitochondrial-dependent pathway in Ehrlich ascites carcinoma tumor

Ursolic acid (UA) is a pentacyclic triterpene naturally occurring in many plant foods. In the present study, we investigated anti-cancer activity of UA in vivo in Ehrlich ascites carcinoma (EAC) tumor. 15 × 10⁶ EAC cells were implanted intraperitoneally (i.p., ascitic tumor) and subcutaneous (s.c., solid tumor) in Swiss albino mice. Mice with established tumors received UA i.p. at 25, 50 and 100 mg/kg bw for 14 d in ascitic and 100 mg/kg bw in solid tumor for 30 d. On day 15, blood samples were collected for hematological assessment of hemoglobin (Hb%), RBCs, WBCs and PCV. Tumor volume, cell viability, angiogenic, anti-angiogenic, anti-inflammatory factors and antioxidant parameters were determined. Immunohistochemistry analysis for VEGF, iNOS, CD31, caspase-3 and Bax were also performed. UA significantly inhibited tumor growth, cell viability, in both ascites and solid tumor model in vivo (p < 0·001). The anti-angiogenic effects were accompanied with decreased VEGF, iNOS, TNF-α and increased IL-12 levels. UA at 100 mg/kg bw dose significantly increased SOD and CAT activity (p < 0.01). GSH and TBARS were increased as compared to control group (p < 0.001). Furthermore, UA increased total RBCs, WBCs as well as Hb% significantly (p < 0.05) compared to cyclophosphamide (CP). Histopathological examination of tumor cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. Decreased peritoneal angiogenesis showed the anti-angiogenic potential. UA downregulated VEGF & iNOS expression whereas bax and caspase-3 expressions were upregulated suggesting drug induced tumor cell apoptosis through activating the pro-apoptotic bcl-2 family and caspase-3 and downregulation of VEGF. The present study sheds light on the potent antitumor property of the UA and can be extended further to develop therapeutic protocols for treatment of cancer.     

Down-regulation of TSG101 by small interfering RNA inhibits the proliferation of breast cancer cells through the MAPK/ERK signal pathway

We designed to investigate the effects of down-regulating the tumor susceptibility gene 101 (TSG101) on the proliferation and apoptosis of the human breast cancer MCF-7 cell line, and the role of the MAPK/ERK signal pathway in this process. The siRNA against TSG101 was transfected into the breast cancer MCF-7 cell line using Lipofectamine 2000. After TSG101 knockdown, the proliferation of MCF-7 cells was measured by the MTT assay. The cell cycle distribution and apoptosis were examined by using flow cytometry while cell migration was measured using a transwell assay. The protein level of p-ERK was further assessed by immunofluorescence and western blotting. Our results are as following, the MCF-7 cells transfected with TSG101 siRNA proliferated significantly slower and exhibited significantly increased rates of apoptosis compared to the control cells. In the TSG101 siRNA transfected cells, the percentage of cells in the G?/G? and S phase of the cell cycle was significantly higher and lower, respectively, compared to the control cells. Moreover, the migration ability of TSG101 siRNA transfected cells was lower than the control groups. Lastly, the level of p-ERK protein in TSG101 siRNA transfected cells was significantly decreased compared with the control cells. In conclusion, TSG101 knockdown in breast cancer cells induces apoptosis and inhibits proliferation. The TSG101 depleted cells are arrested at the G?/S transition of the cell cycle. The migration of breast cancer cells is also impaired by TSG101 siRNA. TSG101 may play a biological role through modulation of the MAPK/ERK signaling pathway in breast cancer.     

Pyran-2-one derivatives from Croton crassifolius as potent apoptosis inducers in HepG2 cells via p53-mediated Ras/Raf/ERK pathway

Chemical investigation of the roots of Croton crassifolius led to the isolation of five pyran-2-one derivatives, including two brand new compounds (1–2), one new natural product (3) and two known compounds (4–5). Their structures and absolute configurations were established by spectroscopic analyses as well as comparison between the calculated optical rotation (OR) values with the experimental data. Interestingly, the new compound 1 showed an unusual negative chemical shift at H-11. It is well known that negative chemical shift values of ¹H NMR spectrum are extremely rare in natural products. Such a negative chemical shift of ¹H NMR spectrum was reproduced by density functional theory (DFT) calculations and explained by the shielding effect from the pyran-2-one ring over the hydrogen atom in the 3D conformations. Then, MTT assay was applied to evaluate the cytotoxicity of the isolated compounds (1–5) against two liver cancer cell lines (HepG2 and MHCC97H). The results suggested that compound 1 displayed the highest cytotoxicity with an IC₅₀ value of 9.8 μM against HepG2 cells. Moreover, there was no obvious cytotoxicity of compounds 1–5 on normal liver cell line LO2. Furthermore, the mechanism of apoptosis induction in compound 1-treated HepG2 cells was investigated. The results showed that compound 1 could induce apoptosis via p53-mediated Ras/Raf/ERK suppression in HepG2 cells.     

Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice

Background

Levamisole, an imidazo(2,1-b)thiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4′-fluorophenyl)-5-thiocyanato-imidazo[2,1-b][1], [3], [4]thiadiazole).

Materials and Methods

ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo.

Results

We have determined the IC₅₀ value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC₅₀ 5 µM). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models.

Conclusion

Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.     

Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.     

BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via downregulating AKT pathway

In our previous study, we demonstrated that the BRCC2 (breast cancer cell 2) gene is a proapoptotic molecule that interacts with Bcl-X_L. BRCC2 downregulation is associated with poor disease-free and overall survival in breast cancer. In this study, we aimed to investigate the role of BRCC2 in tumor suppression in breast cancer. In clinical breast cancer samples, we found that BRCC2 expression was significantly downregulated in cancer lesions compared with paired normal breast tissues. By silencing or overexpressing BRCC2 in breast cancer cells, we found that BRCC2 could inhibit cell growth and metastasis in vitro. An in vivo assay showed that BRCC2 not only dramatically inhibited breast cancer cell xenograft formation and growth but also inhibited breast cancer cell metastasis in a lung metastasis model. Moreover, we demonstrated that BRCC2 inhibited breast cancer metastasis via regulation of the Akt pathway. Thus, our study provided evidence that BRCC2 functions as a novel tumor suppressor in breast cancer and may be a potential therapeutic target for breast cancer management.     

Hypoxia inhibits protein synthesis through a 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer cells

Hypoxia is a state of low oxygen availability that limits tumor growth. The mechanism of protein synthesis inhibition by hypoxia and its circumvention by transformation are not well understood. Hypoxic breast epithelial cells are shown to downregulate protein synthesis by inhibition of the kinase mTOR, which suppresses mRNA translation through a novel mechanism mitigated in transformed cells: disruption of proteasome-targeted degradation of eukaryotic elongation factor 2 (eEF2) kinase and activation of the regulatory protein 4E-BP1. In transformed breast epithelial cells under hypoxia, the mTOR and S6 kinases are constitutively activated and the mTOR negative regulator tuberous sclerosis complex 2 (TSC2) protein fails to function. Gene silencing of 4E-BP1 and eEF2 kinase or TSC2 confers resistance to hypoxia inhibition of protein synthesis in immortalized breast epithelial cells. Breast cancer cells therefore acquire resistance to hypoxia by uncoupling oxygen-responsive signaling pathways from mTOR function, eliminating inhibition of protein synthesis mediated by 4E-BP1 and eEF2.     

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.     

Fhit protein inhibits cell growth by attenuating the signaling mediated by nuclear factor-kappaB in colon cancer cell lines

Fragile histidine triad (FHIT) gene is involved in the deletions at the 3p14.2 region in various cancers. We investigated the role of Fhit protein in cell growth by examining the signaling pathway affected by Fhit. We used 3 human colon cancer cell lines, SW480, DLD-1 and COLO201, in the study. SW480 cells, in which the expression of Fhit is completely absent, were transfected with pIRES1neo vector (SW/IRES cells), wild-type FHIT vector (SW/FHIT cells) or mt-FHIT (codon 96, His changed to Asn) vector (SW/mt-FHIT cells). The growth of SW/FHIT or SW/mt-FHIT cells was suppressed in comparison with that of parent or SW/IRES cells. Especially, the growth of SW/FHIT cells was considerably suppressed. On the other hand, the silencing of FHIT by an siRNA for it in SW/FHIT or DLD-1 cells harboring Fhit demonstrated that the growth of FHIT siRNA-treated cells was significantly enhanced in comparison with that of the vector control or nonspecific siRNA control. Thus, we found that Fhit negatively contributed to cell growth in the colon cancer cell lines. Moreover, SW/FHIT cells exhibited a higher sensitivity to oxidative stress evoked by inhibitors of mitochondrial electron transport or proteasomes compared with any of the control transfectants. The base line amount of phospho-IkappaB-alpha (p-IkappaB-alpha) was reduced in SW/FHIT cells compared with that in the other transfectants. On the contrary, the FHIT siRNA-treated SW/FHIT and DLD-1 cells exhibited an elevated p-IkappaB-alpha level in an RNAi experiment on FHIT. Perturbation of nuclear factor (NF)-kappaB signaling was strongly suggested by the fact that the wild-type Fhit expressants of SW480 cells tended to be sensitive to sulfasarazine or parthenolide, which are inhibitors of NF-kappaB. The time course of the level of IkappaB kinase (IKK) complex (IKKalpha/beta, phospho-IKKalpha/beta and IKKgamma) after the treatment with TNF-alpha was similar between the transfectants. Although p-IkappaB-alpha and phospho-NF-kappaB p65 (p-NF-kappaB) in SW/FHIT cells responded to TNF-alpha as those in other transfectants, the increase in the levels of p-IkappaB-alpha and p-NF-kappaB after a 5-min treatment was less in SW/FHIT cells than in the other transfectants. These results altogether suggest that Fhit functions as an anti-oncoprotein by inhibiting the phosphorylation of IkappaB-alpha and thereby blocking NF-kappaB signaling.     

Valproic acid sensitizes breast cancer cells to hydroxyurea through inhibiting RPA2 hyperphosphorylation-mediated DNA repair pathway

It was reported that valproic acid (VPA, a histone deacetylase inhibitor) can sensitize cancer cells to hydroxyurea (HU, a ribonucleotide reductase inhibitor) for chemotherapy, although the mechanism of VPA-induced HU sensitization is unclear. In this study, we systematically characterized VPA-induced HU sensitization of breast cancer cells. Multiple breast cancer cell models were employed to investigate whether the safe concentration of 0.5 mM VPA and 2 mM HU can result in DNA double-strand breaks (DSBs) and impact cell survival. Furthermore, the underlying mechanism was explored through cell biology assays, including clonogenic survival, homologous recombination (HR) activity, immunoblot and immunofluorescence. We found that VPA and HU cooperatively suppressed cancer cell survival. VPA resulted in the accumulation of more DNA double-strand breaks (DSBs) in response to HU-induced replication arrest and was able to block HU-stimulated homologous recombination (HR) through inhibiting the activity of two key HR repair proteins by hyperphosphorylation of replication protein A2 (RPA2-p) and recombinase Rad51. However, apoptosis was not detected under this condition. In addition, the results from the survival fraction in the cells expressing defective RPA2-p showed that VPA disrupted the HU-induced RPA2-p-Rad51-mediated HR pathway. Importantly, these findings were further supported by analyzing primary-culture cells from the tissue of chemical carcinogen (DMBA)-induced breast cancer in rats. Thus, our data demonstrated that VPA and HU synergistically suppressed tumor cells via disturbing RPA2-p-mediated DNA repair pathway, which provides a new way for combining chemotherapeutic drugs to sensitize breast cancer cells.