(-)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.     

(-)-Epigallocatechin gallate can prevent cisplatin-induced lung tumorigenesis in A/J mice

Risks of secondary lung cancer in patients with non-small cell lung cancer and small cell lung cancer are estimated to be 1-2% and 2-10% per patient per year, respectively. Cisplatin is widely used in the treatment of lung cancer and is also known as a carcinogen in experimental animals. In this study, the effect of (-)-epigallocatechin gallate (EGCG) on cisplatin-induced lung tumors in A/J mice was investigated. Female A/J mice (4 weeks old) were divided into four groups: group 1, control without treatment; group 2, EGCG treatment (1 mg/ml in tap water); group 3, weekly cisplatin treatment (1.62 mg/kg body wt, i.p.) for 10 weeks; group 4, cisplatin plus EGCG treatment (EGCG was started 2 weeks before cisplatin treatment). Four groups of mice were killed at week 30 after treatment. Tumor incidence was 26.3% (5/19) in group 1, 30% (6/20) in group 2, 100% (19/19) in group 3 and 94.4% (17/18) in group 4. Tumor multiplicity (the number of tumors per mouse, mean +/- SD) was 0.4 +/- 0.8 in group 1, 0.4 +/- 0.8 in group 2, 5.1 +/- 2.1 in group 3 and 2.8 +/- 2.3 in group 4. Tumor multiplicity was significantly reduced by adding EGCG to cisplatin-treated mice (P < 0.01). Furthermore, EGCG significantly reduced cisplatin-induced weight loss from 24.7-26.3% (cisplatin treatment) to 10.8-11.6% (cisplatin plus EGCG treatment) (P < 0.01). These findings suggest that EGCG can inhibit cisplatin-induced weight loss and lung tumorigenesis in A/J mice.     

Maspin sensitizes breast carcinoma cells to induced apoptosis

Maspin, a novel serine protease inhibitor (serpin), suppresses the growth and metastasis of breast tumor in vivo. However, the underlying molecular mechanism is unclear. In the current study, we report the first evidence that endogenous maspin expression in mammary carcinoma cells MDA-MB-435 enhanced staurosporine (STS)-induced apoptosis as judged by the increased fragmentation of DNA, increased proteolytic inactivation of poly-[ADP-ribose]-polymerase (PARP), as well as the increased activation of caspase-8 and caspase-3. In parallel, recombinant maspin did not directly regulate the proteolytic activities of either caspase-3 or caspase-8 in vitro. Consistent with this result, maspin expressing normal mammary epithelial cells underwent more rapid STS-induced apoptosis as compared to breast carcinoma cells. Interestingly, maspin transfectant cells did not undergo spontaneous apoptosis in the absence of STS. Moreover, neither purified maspin protein added from outside nor endogenous maspin secreted to the cell culture media sensitized cells to STS-induced apoptosis. To investigate the structural determinants of maspin in its apoptosis-sensitizing effect, MDA-MB-435 cells were also transfected with maspin/PAI-1 and PAI-1/maspin chimeric constructs resulting from swapping the N-terminal and the C-terminal domains between maspin and PAI-1 (plasminogen activator inhibitor type 1). The resulting stable transfectant clones expressing maspin/PAI-1 and PAI-1/maspin, respectively, did not undergo spontaneous apoptosis, and were similarly inhibited as maspin transfectant cells in motility assay. Interestingly, however, expression of both maspin/PAI-1 and PAI-1/maspin in MDA-MB-435 cells failed to sensitize these cells to STS-induced apoptosis. Taken together, our evidence provides new insights into the complex molecular mechanisms of maspin that may suppress breast tumor progression not only at the step of invasion and motility, but also by regulating tumor cell apoptosis. The sensitizing effect of maspin on apoptosis is to be contrasted by the pro-survival effect of several other serpins.     

Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin,paclitaxel and cyclophosphamide

Overexpression of the anti-apoptotic proteins bcl-2 and bcl-xL is implicated in breast cancer development, tumor progression and drug resistance. Here we describe the use of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 to sensitize breast carcinoma cells to anti-cancer drugs routinely used in breast cancer therapy. MCF7 cells were treated with oligonucleotide 4625, doxorubicin, paclitaxel or cyclophosphamide alone, or with combinations of oligonucleotide and the anti-cancer drugs. As measured in cell viability assays, treatment with the various combinations reduced the number of viable MCF7 cells more effectively than treatment with the single drugs alone. Treatment with a sequence control oligonucleotide did not affect cell viability. All combination treatments induced apoptosis as demonstrated by the appearance of massive nuclear condensation in a high proportion of the cells. To further characterize the interaction between 4625 and doxorubicin, paclitaxel or cyclophosphamide, the median-effect method was used. In MCF7 cells all combinations resulted in potent synergistic effects over a broad range of toxicity with combination indices ranging from 0.8 to 0.1. Similarly, strong synergistic interactions between oligonucleotide 4625 and the anti-cancer drugs were also observed in cultures of the breast carcinoma cell line MDA-MB-231. Our data suggest the use of 4625 as a potent adjuvant in breast cancer chemotherapy.     

Letrozole sensitizes breast cancer cells to ionizing radiation

Introduction  

Radiotherapy (RT) is considered a standard treatment option after surgery for breast cancer. Letrozole, an aromatase inhibitor, is being evaluated in the adjuvant setting. We determined the effects of the combination of RT and letrozole in the aromatase-expressing breast tumour cell line MCF-7CA, stably transfected with the CYP19 gene.     

(-)-Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts

Bilateral synchronous breast cancer appears to have a worse prognosis than comparable unilateral breast cancer. HER-2/neu expression in bilateral breast cancer has not been reported. The purpose of this study was to review the characteristics of patients with bilateral synchronous breast cancer and to report the incidence of HER-2/neu overexpression. Between 1984 and 1998, 58 patients were diagnosed with bilateral synchronous breast cancer (defined as both cancers diagnosed within 3 months). The paraffin blocks from both breast specimens were available and immunostained in 21 patients. Of 42 breast specimens, there were 31 invasive carcinomas and 11 noninvasive carcinomas. Of the 21 paired specimens immunostained for HER-2/neu, 11 were invasive cancers in both breasts, nine were invasive cancers in one breast and noninvasive cancers in the other breast, and one was noninvasive cancers in both breasts. Of the 31 invasive carcinomas, HER-2/neu was overexpressed (2–3+) in 22 (71%) and negative (0–1+) in nine (29%). In contrast, 35 of 101 (34.7%) consecutive unilateral invasive breast cancer specimens from our institution overexpressed HER-2/neu. The difference in HER-2/neu overexpression between patients with bilateral synchronous breast cancer and unilateral breast cancer (22/31 v.s. 35/101) was statistically significant (chi square = 11.3, p   <  0.001). In cases where both breasts had invasive carcinoma, HER-2/neu overexpression could be either in one (six patients) or both breasts (four patients). The increased mortality of patients with bilateral synchronous breast cancer may be due to the higher incidence of HER-2/neu overexpression.     

S100P sensitizes ovarian cancer cells to carboplatin and paclitaxel in vitro

Objective

To investigate the relationship between the S100P and the sensitivity of ovarian cancer to chemotherapeutics.

Method

We established stable cell lines of ovarian cancer cells, SKOV3 and OVCAR3, that overexpress human S100P. We also transiently transfected the parent cell lines with S100P-targeted siRNA for down-regulation of S100P expression. The sensitivity of all transfected and untransfected cell lines to carboplatin and paclitaxel was detected by MTT assay.

Results

For both cells, IC_50s decreased to carboplatin and paclitaxel (p < 0.05), with overexpression of S100P compared to untransfected cells. Alternatively, with down-regulation of S100P by siRNA, the IC₅₀ to carboplatin and paclitaxel increased in each case (p < 0.05), which was significantly higher compared to untransfected cells.

Conclusion

Changes in expression levels of S100P in SKOV3 and OVCAR3 cells results in variable susceptibility to carboplatin and paclitaxel. These data suggest that S100P contributes to chemosensitivity to carboplatin and paclitaxel in ovarian cancer cells.     

MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo

Nasopharyngeal carcinoma (NPC) is an endemic tumor with a relatively high incidence in Southern China and Southeast Asia. Paclitaxel combination chemotherapy has been used for treatment of advanced NPC. However, treatment failure often occurs due to development of acquired paclitaxel resistance. In this study, we first established a paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cell sublines by treating the parental CNE-1, HNE-2 and 5–8F cells with increasing doses of paclitaxel for about 5 months, respectively. Then, microRNA arrays were used to screen differentially expressed miRNAs between the CNE-1/Taxol cells and the parental CNE-1 cells. We found 13 differentially expressed miRNAs, of which miR-1204 was significantly downregulated in the paclitaxel-resistant CNE-1/Taxol cells. We restored miR-1204 expression in the CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells and found that restoration of miR-1204 re-sensitized the paclitaxel-resistant CNE-1/Taxol, HNE-2/Taxol and 5–8F/Taxol cells to paclitaxel both in vitro. Finally, we demonstrated that restoration of miR-1204 in significantly inhibits tumor growth in vivo. Thus, our study provides important information for the development of targeted gene therapy for reversing paclitaxel resistance in NPC.     

(-)-Epigallocatechin gallate overcomes resistance to etoposide-induced cell death by targeting the molecular chaperone glucose-regulated protein 78

Many beneficial properties have been attributed to (-)-epigallocatechin gallate (EGCG), including chemopreventive, anticarcinogenic, and antioxidant actions. In this study, we investigated the effects of EGCG on the function of glucose-regulated protein 78 (GRP78), which is associated with the multidrug resistance phenotype of many types of cancer cells. Our investigation was directed at elucidating the mechanism of the EGCG and GRP78 interaction and providing evidence about whether EGCG modulates the activity of anticancer drugs through the inhibition of GRP78 function. We found that EGCG directly interacted with GRP78 at the ATP-binding site of protein and regulated its function by competing with ATP binding, resulting in the inhibition of ATPase activity. EGCG binding caused the conversion of GRP78 from its active monomer to the inactive dimer and oligomer forms. Further, we showed that EGCG interfered with the formation of the antiapoptotic GRP78-caspase-7 complex, which resulted in an increased etoposide-induced apoptosis in cancer cells. We also showed that EGCG significantly suppressed the transformed phenotype of breast cancer cells treated with etoposide. Overall, these results strongly suggested that EGCG could prevent the antiapoptotic effect of GRP78, which usually suppresses the caspase-mediated cell death pathways in drug-treated cancer cells, contributing to the development of drug resistance.     

Inhibition of metadherin sensitizes breast cancer cells to AZD6244

The development of systemic therapy drug resistance for breast cancer treatment is an ongoing problem, thus, so are the potential molecular mechanisms of it. AZD6244 is a novel ATP-uncompetitive inhibitor to MAP/ERK kinase (MEK) 1/2 which has been demonstrated to be potent, selective and safe in the clinical trials and previous studies. However, the precise role of resistance to AZD6244 is largely unknown. We and other groups have reported that the novel oncogene Metadherin (MTDH) is associated with multiple drug resistance, but there is no report about its role in treatment of AZD6244. Here we report that the resistance to AZD6244 can be reserved by downregulating MTDH in breast cancer cell lines. When the MTDH was downregulated, the breast cancer cells exhibited a significantly increased sensitivity to AZD6244 as measured by MTT assay. After treated with AZD6244 the MTDH-knockdown cells showed more apoptosis rate and growth inhibition. We also showed that knockdown of MTDH cannot only increase expression of FOXO3a but also activate it by promoting its translocation via MTDH/ERK1/2/FOXO3a pathway rather than MTDH/AKT/FOXO3a pathway. In conclusion knockdown MTDH can enhance the breast cancer cells sensitivity to AZD6244 via regulating the expression and activity of FOXO3a. These indicate us that MTDH is a candidate marker to predict the clinical efficacy of AZD6244 and targeting MTDH could overcome the resistance to AZD6244 in breast cancer cells.     

Rad51 inhibition sensitizes breast cancer stem cells to PARP inhibitor in triple-negative breast cancer

Breast cancer susceptibility gene 1 (BRCA1) is a tumorsuppressor gene, and its protein BRCA1 plays a role inDNA repair [1]. BRCA1 is generally expressed in the cellsof mammary glands and other tissues, helping to repairdamaged DNA or disrupting cells when DNA cannot berepaired. When BRCA1 is mutated and cannot functionand therefore the damaged DNA cannot be repaired ontime, the risk of breast cancer will greatly increase [2]. InBRCA1-mutant tumors, the capability of DNA damagerepair is decreased, which makes tumor cells sensitiveto DNA-damaging drugs; however, high BRCA1 activityweakens the effect of these drugs [3].     

(-)-Epigallocatechin gallate inhibits membrane-type 1 matrix metalloproteinase, MT1-MMP, and tumor angiogenesis

Membrane-type 1 matrix metalloproteinase (MT1-MMP), which hydrolyzes type I collagen and activates MMP-2, are deeply involved in angiogenesis as well as in tumor cell invasion and metastasis. We previously screened a number of natural and synthetic compounds to obtain a specific inhibitor of MT1-MMP and observed that (-)-epigallocatechin gallate (EGCG) has a potent and distinct inhibitory activity against MT1-MMP. In the present study, we investigated the effect of EGCG on tumor angiogenesis. EGCG significantly inhibited the invasion of human umbilical vein endothelial cells (HUVECs) at the concentration of 10 microM. This effect was not due to the toxicity of EGCG since this concentration of EGCG did not affect the HUVEC growth. Furthermore, morphological change of HUVEC at this concentration of EGCG was not observed under confocal laser scanning microscopy. EGCG suppressed tube formation by HUVECs in vitro and angiogenesis in vivo by using dorsal air sac model. Finally, we observed that both colon 26 NL17 carcinoma and Meth A sarcoma growth was suppressed in these tumor-bearing mice by EGCG administration, at least partly though the inhibition of angiogenesis.     

High energy shock waves (HESW) increase paclitaxel efficacy in a syngeneic model of breast cancer

The combined effect of high energy shock waves (HESW), generated by a piezoelectric device, and paclitaxel on Mat B-III rat breast cancer cells in vitro and in an in vivo animal model is presented. A significant reduction of in vitro Mat B-III cell proliferation versus cells treated with paclitaxel alone was observed with the combined exposure to paclitaxel (0.1, 1, or 10 nM) and HESW (0.22 mJ/mm2, 1000 shots). Moreover earlier induction and enhanced apoptosis occurred in cells subjected to the combined treatment with paclitaxel and HESW at 1 and 10 nM versus paclitaxel alone (p<0.01 and p<0.001, respectively). The percentage of apoptotic cells along with BAD mRNA expression, confirm a significant enhancement of apoptosis in tumor tissues subjected to the combined treatment with paclitaxel (2.5 mg/kg on days 7 and 11) and HESW (0.50 mJ/mm2, 500 shots on day 11) in comparison with paclitaxel alone. In conclusion, these data suggest that HESW enhance paclitaxel cytotoxicity in the Mat B-III syngeneic model of breast cancer.     

Silencing of CXCR4 sensitizes triple-negative breast cancer cells to cisplatin

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer for which there is no effective treatment. Previously, we and others demonstrated that CXCR4 surface expression is an independent prognostic factor for disease relapse and survival in breast cancer. In this study, we investigated the effects of CXCR4 gene silencing on cisplatin chemosensitivity in human triple-negative breast cancer cell lines. We found that CXCR4 silencing significantly inhibited cell growth, decreased colony formation, and enhanced cisplatin sensitivity while overexpression of CXCR4 rendered cells more resistant to cisplatin. Moreover, the percentage of apoptosis and cell cycle arrest at the G2/M phase of cisplatin-treated CXCR4 knockdown cells was significantly higher than control cells. Furthermore, we demonstrated CXCR4 knockdown cells showed lower levels of mutant p53 and Bcl-2 protein than the control group, while also having higher levels of caspase-3 and Bax. However overexpression of CXCR4 had the reverse effect. In vivo experiments confirmed that downregulation of CXCR4 enhanced cisplatin anticancer activity in tumor-bearing mice, and that this enhanced anticancer activity is attributable to tumor cell apoptosis. Thus, this study indicates that CXCR4 can modulate cisplatin sensitivity in TNBC cells and suggests that CXCR4 may be a therapeutic target for TNBC.     

Thrombin-cleaved COOH(-) terminal osteopontin peptide binds with cyclophilin C to CD147 in murine breast cancer cells

Osteopontin is a glycoprotein that has been linked to metastatic function in breast, lung, and prostate cancers. However, the mechanism by which osteopontin acts to induce metastatic properties is largely unknown. One intriguing feature of osteopontin is the presence of a conserved thrombin cleavage site that is COOH-terminal from a well-characterized RGD domain. Although the COOH-terminal fragment may bind to cell surface CD44 receptors, little is known about the COOH-terminal osteopontin fragment. In the current study, we use the murine mammary epithelial tumor cell lines 4T1 and 4T07; these cells are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. Using flow cytometry and Forster resonance energy transfer, we show that the COOH-terminal fragment of osteopontin binds with another marker of metastatic function (cyclophilin C or rotamase) to the CD147 cell surface glycoprotein (also known as extracellular matrix metalloproteinase inducer), to activate Akt1/2 and matrix metalloproteinase-2. In in vitro assays, thrombin cleavage of osteopontin to generate short COOH-terminal osteopontin in the presence of cyclophilin C increases migration and invasion of both 4T07 and 4T1 cells. This interaction between osteopontin peptide and cyclophilin C has not been previously described but assigns a heretofore unknown function for the thrombin-cleaved osteopontin COOH-terminal fragment.     

Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis

Introduction  

Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.     

2-methoxyestradiol sensitizes breast cancer cells to taxanes by targeting centrosomes

Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC₅₀ values in all cells tested ranging from 28–44% (P < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; P < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; P < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

Cyclin d1 overexpression sensitizes breast cancer cells to fenretinide.

PURPOSE: Fenretinide has shown promise in the chemoprevention of breast cancer, a tumor type in which the oncogene cyclin D1 is overexpressed frequently. We aimed at determining the effect of cyclin D1 level on the response to fenretinide treatment. EXPERIMENTAL DESIGN: Stable clones of T47-D cells were created to overexpress cyclin D1 or a mutant of cyclin D1, injected in nude mice for xenograft formation, and the rate of tumor growth and tumor regression determined. RESULTS: We show here that cells overexpressing cyclin D1 are significantly more sensitive to fenretinide than genetically matched cells that express low levels of cyclin D1, and that fenretinide prevents tumor formation arising from cyclin D1-overexpressing cells. Furthermore, we show that fenretinide is also able to promote the regression of cyclin D1-positive tumors. We also show that cells expressing a mutant of cyclin D1 that cannot bind to cdk4 are also more sensitive to fenretinide. CONCLUSIONS: These results suggest that fenretinide may be particularly useful in the treatment of cyclin D1-positive breast cancers, and that the interaction between cyclin D1 and fenretinide is independent of cyclin D1 binding to cdk4.