Bcl-2 Gene Product Research Articles

Protein Expression Profiling Identifies Key Proteins and Pathways Involved in Growth Inhibitory Effects Exerted by Guggulsterone in Human Colorectal Cancer Cells.

Colorectal cancer (CRC) is a leading killer cancer worldwide and one of the most common malignancies with increasing incidences of mortality. Guggulsterone (GS) is a plant sterol used for treatment of various ailments such as obesity, hyperlipidemia, diabetes, and arthritis. In the current study, anti-cancer effects of GS in human colorectal cancer cell line HCT 116 was tested, potential targets identified using mass spectrometry-based label-free shotgun proteomics approach and key pathways validated by proteome profiler antibody arrays. Comprehensive proteomic profiling identified 14 proteins as significantly dysregulated. Proteins involved in cell proliferation/migration, tumorigenesis, cell growth, metabolism, and DNA replication were downregulated while the protein with functional role in exocytosis/tumor suppression was found to be upregulated. Our study evidenced that GS treatment altered expression of Bcl-2 mediated the mitochondrial release of cytochrome c which triggered the formation of apoptosome as well as activation of caspase-3/7 leading to death of HCT 116 cells via intrinsic apoptosis pathway. GS treatment also induced expression of p53 protein while p21 expression was unaltered with no cell cycle arrest. In addition, GS was found to inhibit NF-kB signaling in colon cancer cells by quelling the expression of its regulated gene products Bcl-2, cIAP-1, and survivin.

Effect of recombinant human endostatin on hypertrophic scar fibroblast apoptosis in a rabbit ear model

Hypertrophic scar (HS) is a dermal fibroproliferative disorder characterized by the excessive proliferation of fibroblasts and is thought to result from a cellular imbalance caused by the increased growth and reduced apoptosis of hypertrophic scar fibroblasts (HSFs). Our recent study demonstrated that recombinant human endostatin (rhEndostatin) plays a key role in the inhibition of HSF proliferation in vitro, with a resulting decrease in dermal thickness and scar hypertrophy. However, the effect of this protein on HSF apoptosis is unknown. The present study was undertaken to directly examine the effect of rhEndostatin on HSF apoptosis in the rabbit ear model. Transmission electron microscopy and flow cytometry were used to investigate HSF apoptosis in scar tissues and cultured HSFs in vitro, respectively. The expression levels of the c-jun, c-fos, NF-κB, fas, caspase-3, and bcl-2 gene products in HSFs were quantified using real-time PCR and Western blotting assays. Our data reveal that rhEndostatin (2.5 or 5mg/ml) induces HSF apoptotic cell death in scar tissue. Additionally, HSFs treated with rhEndostatin (100mg/L) in vitro accumulated in early and late apoptosis and displayed significantly decreased expression of c-jun, c-fos, NF-κB, fas, caspase-3 and bcl-2. In sum, these results demonstrate that rhEndostatin induces HSF apoptosis, and this phenotypeis partially due to downregulation of NF-κB and bcl-2. These findings suggest that rhEndostatin may have an inhibitory effect on scar hypertrophy in vivo via HSF apoptotic induction and therefore has potential therapeutic use for the treatment of HS.

Gene expression, oocyte quality and embryo production by cloning in goats supplemented with different diets

The goal of this study was to assess the effect of using native food sources (cashew apple bagasse (Anacardium occidentale), cassava root scrapings (Manihot esculenta), and wax palm fruit (Copernicia prunifera)) as corn replacement in the balanced feed of goats as oocyte donors for cloning procedures. After four weeks of feeding, we evaluated follicular development and fluid composition, in vivo maturation rate and oocyte competence assessed by in vitro embryo production by cloning, and the expression pattern of GDF9, BMP15, BAX, and BCL2 gene products in oocytes. Type of feed had no effect on the live weight and sternal subcutaneous fat thickness in goats, measured prior to oocyte harvesting. Inclusion of wax palm fruit in the foodstuff significantly increased the number of aspirated follicles, number of recovered oocytes, and glucose concentrations in the follicular fluid. However, maturation rates and in vitro embryo yield were not affected by the type of foodstuff. Our results also showed that the test diets induced underexpression of GDF9 and did not affect oocyte developmental capacity or the efficiency of the cloning process under our experimental conditions. In general, inclusion of alternative foodstuff in the diet of adult oocyte donor goats as replacement for corn was effective in maintaining oocyte developmental capacity, being as successful in supporting in vitro embryo production by cloning procedures in goats. The alternative food ingredients used for a period of four weeks can effectively replace corn in the diet without appearing to compromise animal health and fertility, which is an economic and viable alternative for goat production in tropical regions, contributing to the current trend in global sustainability.

Adenosine kinase expression in cortical dysplasia with balloon cells: analysis of developmental lineage of cell types.

Focal cortical dysplasia type IIB (FCDIIB) is a developmental malformation of the cerebral cortex that is associated with pharmacoresistant epilepsy. Overexpression of adenosine kinase (ADK) has been regarded as a pathologic hallmark of epilepsy. We hypothesized that the epileptogenic mechanisms underlying FCDIIB are related to abnormal ADK expression. We used immunohistochemistry to examine the expression of ADK and of heterogeneous cell population markers of astrocytes (glial fibrillary acidic protein), immature glia (vimentin), immature neurons (neuronal class III beta-tubulin, TUJ1), multipotential progenitor cells (nestin), mature neurons (microtubule-associated protein 2), and antiapoptotic gene products (Bcl-2) in surgically resected human epileptic cortical specimens from FCDIIB patients (n = 20). Expression patterns were compared with those in normal autopsy (n = 6) and surgical control (n = 6) brain samples. Balloon cells in FCDII lesions were immunoreactive for ADK (77%) and balloon cells expressing the different cell markers expressing different degrees of ADK. Adenosine kinase expression assessed by Western blot and enzymatic activity were also greater in FCD versus control samples. These results suggest that upregulation of ADK is a common pathologic component of FCDIIB. Adenosine kinase might, therefore, be a target in the treatment of epilepsy associated with FCD.

Early methyl donor deficiency may induce persistent brain defects by reducing Stat3 signaling targeted by miR-124

The methyl donors folate (vitamin B9) and vitamin B12 are centrepieces of the one-carbon metabolism that has a key role in transmethylation reactions, and thus in epigenetic and epigenomic regulations. Low dietary intakes of folate and vitamin B12 are frequent, especially in pregnant women and in the elderly, and deficiency constitutes a risk factor for various diseases, including neurological and developmental disorders. In this respect, both vitamins are essential for normal brain development, and have a role in neuroplasticity and in the maintenance of neuronal integrity. The consequences of a methyl donor deficiency (MDD) were studied both in vivo in rats exposed in utero, and in vitro in hippocampal progenitors (H19-7 cell line). Deficiency was associated with growth retardation at embryonic day 20 (E20) and postnatally with long-term brain defects in selective areas. mRNA and protein levels of the transcription factor Stat3 were found to be decreased in the brains of deprived fetuses and in differentiating progenitors (62 and 48% for total Stat3 protein, respectively), along with a strong reduction in its phosphorylation at both Tyr705 and Ser727 residues. Vitamin shortage also affected upstream kinases of Stat3 signaling pathway (phospho-Erk1/2, phospho-Src, phospho-JNK, and phospho-p38) as well as downstream target gene products (Bcl-2 and Bcl-xL), thus promoting apoptosis. Conversely, the expression of the Stat3 regulator miR-124 was upregulated in deficiency conditions (≥65%), and its silencing by using siRNA partly restored Stat3 signaling in hippocampal neurons by increasing specifically the phosphorylation of Erk1/2 and Src kinases. Furthermore, miR-124 siRNA improved the phenotype of deprived cells, with enhanced neurite outgrowth. Taken together, our data suggest that downregulation of Stat3 signaling by miR-124 would be a key factor in the deleterious effects of MDD on brain development.

Study of immunohistochemical demonstration of Bcl-2 protein in ameloblastoma and keratocystic odontogenic tumor

Background:The Bcl-2 (B-cell lymphoma) gene product also known as apoptotic inhibitor is expressed in many normal and tumor tissues. This Bcl-2 gene protects the cell by blocking postmitotic differentiation from apoptosis, thus maintaining the stem cell pool.Objective:To study the expression of Bcl-2 protein in ameloblastoma and keratocystic odontogenic tumor (KCOT) to determine their apoptotic behaviors and to analyze biological nature of KCOT, which has higher proliferative potential and aggressive clinical behavior like odontogenic tumors.Materials and Methods:Formalin-fixed paraffin sections of ameloblastoma (n = 20) and KCOT (n = 20) are considered for immunohistochemical analysis using monoclonal antibody against antihuman Bcl-2 oncoprotein. Lymphomas (n = 3) were used as controls.Statistical Analysis:The statistical analysis was performed using software package of social science version 16. The data were analyzed using Chi-square test and Student's t test. In all the above tests, P < 0.05 was accepted as statistically significant.Results:The positive ratio of Bcl-2 was 85% (17/20) in ameloblastoma, 85% (17/20) in KCOT and 100% (3/3) in lymphomas. Bcl-2 was expressed in peripheral cells and few scattered cells of stellate reticulum in ameloblastoma. KCOT showed strong positivity for Bcl-2 mainly in the basal layer.Interpretation and Conclusion:The present study demonstrates the aggressive nature of KCOT and intrinsic growth potential of its lining epithelium. This study clearly demonstrates that KCOT like ameloblastoma demonstrates aggressive clinical and noticeable invasive behavior. Therefore, it is now considered as no longer a developmental cyst but as odontogenic tumor.

Abstract S5-8: Parallel upregulation of Bcl2 and estrogen receptor (ER) expression in HER2+ breast cancer patients treated with neoadjuvant lapatinib.

Abstract Background: We previously showed in HER2+ models of breast cancer (BC) that potent inhibition of the HER receptor layer can lead to re-expression of estrogen receptor (ER) or activation of the ER pathway. Consequently, the anti-apoptotic ER gene product Bcl2 is upregulated, resulting in enhanced tumor cell survival and treatment resistance. In this study, we investigated whether Bcl2 and ER expression levels are simultaneously increased by neoadjuvant treatment with the dual HER1/2 tyrosine kinase inhibitor lapatinib in HER2+ BC patients. Methods: In a neoadjuvant phase II clinical trial 49 HER2+ BC patients were treated with lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before surgery. Tumor specimens were prospectively collected at different time-points during lapatinib treatment (baseline, and weeks 2 and 4). Bcl2, ER, progesterone receptor (PR), total (t) and phosphorylated (p)-HER2, and Ki67 were assessed by immunohistochemistry. Spearman correlation was used to evaluate the association among the biomarkers at baseline, and the correlation of their changes over time. Fisher's Exact test and non-parametric Wilcoxon rank sum test were used respectively to determine if the frequency and the magnitude of Bcl2 expression changes were associated with baseline ER status. Results: 35/49 HER2+ tumor specimens (71%) were available for baseline evaluation of Bcl2 and ER. Of those, 12 (34%) were ER-positive (Allred score ≥ 3) and 23 (66%) ER-negative. Baseline Bcl2 expression correlated positively with ER (r = .75; p &amp;lt; .0001) and PR (r = .53; p = .0015), and inversely with t-HER2 (r = −.43; p = .0097). ER baseline expression correlated positively with PR (r = .57; p = .0004), and inversely with t and p-HER2 (r = −.55; p = .0005, and r = −.37; p = .0282, respectively) and Ki67 (r = −.39; p = .0271). Bcl2 changes at week 2 (w2) positively correlated with changes in both ER and PR levels (r = .70; p = .0002 and r = .57; p = .0076, respectively). Additionally, the increase in Bcl2 expression, observed in 9 of the 23 (39%) tumors with tissue available at w2, was significantly more frequent (p = .0147) and of greater magnitude (p = .0001) in ER-pos vs. ER-neg tumors — 8/9 ER-pos tumors at w2 (including 3 converted from ER-neg by lapatinib) had increased Bcl2, while only 1 of the 14 (7%) ER-neg tumors (at baseline and w2) had increased Bcl2. The expression of ER itself at w2 also increased in 3 out of the 6 (50%) tumors which were originally ER-pos and had tissue available at w2, and in all of them Bcl2 increased in parallel. Of note, the single baseline ER-pos tumor that showed a reduction in ER at w2 had a parallel decrease in Bcl2. Similar observations or trends were found at week 4. Conclusion: Our study suggests that Bcl2 is upregulated as a result of enhanced/restored ER activity upon anti-HER2 therapy with lapatinib in HER2+ tumors. This further supports the use of endocrine along with anti-HER-2 therapy to block this escape pathway which could otherwise cause treatment resistance. In addition, the ER re-expression with lapatinib treatment observed in this study emphasizes the need to re-biopsy HER2+/ER− patients receiving anti-HER2 therapy and to add endocrine therapy if the tumor becomes ER-positive. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S5-8.

Bcl-XL, but not Bcl-2, can protect human B-lymphoma cell lines from parthenolide-induced apoptosis

In this report, we investigated the effects of the natural product parthenolide on human B-lymphoma cell lines. We show that parthenolide inhibited NF-κB transcription factor c-Rel (REL). In addition, the sensitivity of several human B-lymphoma cell lines to parthenolide-induced apoptosis inversely correlated with their levels of anti-apoptosis protein Bcl-XL. Furthermore, ectopic expression of Bcl-XL (but not Bcl-2) in two B-lymphoma cell lines decreased their sensitivity to parthenolide-induced apoptosis. Finally, over-expression of a transforming mutant of REL, which increased expression of endogenous Bcl-XL, decreased the sensitivity of BJAB B-lymphoma cells to parthenolide-induced apoptosis. These results demonstrate that the NF-κB target gene products Bcl-XL and Bcl-2 can play different roles in protecting B-lymphoma cells from chemical-induced apoptosis.

Diphenyl Difluoroketone: A Potent Chemotherapy Candidate for Human Hepatocellular Carcinoma

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G2/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB–regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma.

Curcumin reverses chemoresistance of human gastric cancer cells by downregulating the NF-κB transcription factor

Gastric cancer remains one of the major health problems worldwide. Chemotherapy is an important therapeutic modality for gastric cancer, but the success rate of this treatment is limited because of chemoresistance. The ubiquitously expressed transcription factor NF-κB has been suggested to be associated with chemoresistance of gastric cancer. Agents that can either enhance the effects of chemotherapeutics or overcome chemoresistance to chemotherapeutics are needed for the treatment of gastric cancer. Curcumin, a component of turmeric, is one such agent that has been shown to suppress NF-κB and increase the efficacy of chemotherapy. In this study, we investigated whether curcumin can reverse chemoresistance by downregulating NF-κB in human gastric cancer cells. SGC-7901 human gastric cancer cells was treated with chemotherapeutics (etoposide and doxorubicin) or by combined application of curcumin and chemotherapeutics. The viability of SGC-7901 cells was measured by MTT assay. Apoptosis of SGC-7901 cells was detected using the TUNEL and Annexin V/PI methods. The protein levels of NF-κB were analyzed by immunocytochemical staining. EMSA was used to confirm the increased nuclear translocation of RelA. The protein levels of p-IκBα, Bcl-2 and Bcl-xL were analyzed by Western blotting. The chemotherapeutics (etoposide and doxorubicin) suppressed the growth of SGC-7901 cells, in a time-dose-dependent manner. Use of curcumin in addition to these agents can suppress cell growth further (inhibitory rate: doxorubicin vs. doxorubicin + curcumin, 33% vs. 45%, p<0.05; etoposide vs. etoposide + curcumin, 35% vs. 48%, p<0.05). Furthermore, chemotherapeutics induced apoptosis of SGC-7901 cells and activated NF-κB. The combination of curcumin and chemotherapeutics induced apoptosis of SGC-7901 cells further, attenuated the activation of NF-κB, and reduced expression of the NF-κB-regulated anti-apoptotic gene products Bcl-2 and Bcl-xL. Curcumin potentiates the antitumor effects of chemotherapeutics in gastric cancer by suppressing NF-κB and NF-κB-regulated anti-apoptotic genes.

P53 and BCL-2 Over-Expression Inversely Correlates with Histological Differentiation in Occupational Ethmoidal Intestinal-Type Sinonasal Adenocarcinoma

Despite their histological resemblance to colorectal adenocarcinoma, there is little information on the molecular events involved in the pathogenesis of intestinal-type sinonasal adenocarcinoma (ITACs). The aim of this paper is to evaluate the possible role of TP53 and Bcl-2 gene defects in ITAC by investigating the immunohistochemical expression of TP53 and Bcl-2 gene products in a group of ethmoidal ITACs associated with occupational exposure. A retrospective study on 15 patients with pathological diagnosis of primary ethmoidal ITAC was conducted. Representative formalin-fixed, paraffin wax-embedded block from each case was selected for immunohistochemical studies using the antibodies against p53 and Bcl-2. Clinical-pathological data were also correlated with the staining results. The results of immunohistochemical examination demonstrated that poorly differentiated cases showed a higher percentage of p53 and Bcl-2 expressing cells in comparison to well-differentiated cases. No correlation was found with other clinico-pathological parameters, including T, stage and relapses. The relationship between up-regulation of p53 and Bcl-2 and poorly differentiated ethmoidal adenocarcinoma suggests a role of these genes, in combination with additional genetic events, in the pathogenesis of ITAC.

The biological and clinical impact of inhibition of NF‐κB‐initiated apoptosis in diffuse large B cell lymphoma (DLBCL)

NF-κB is frequently over-expressed in a variety of non-Hodgkin's lymphomas (NHLs) and has been implicated in lymphomagenesis; however, its role in diffuse large B cell lymphoma (DLBCL) as a prognostic biomarker has not been fully elucidated. Therefore, we investigated the role of NF-κB and its association with clinicopathological features in a tissue microarray cohort of 230 DLBCL patient samples. We then elucidated the role of NF-κB inhibition on cell viability and apoptosis in vitro, using DLBCL cell lines. Using immunohistochemistry, NF-κB was detected in 25.6% (52/203) DLBCL tumours, was associated with activated B cell (ABC) phenotype (p = 0.0054), Epstein-Barr virus (EBV; p = 0.0080) and over-expression of the anti-apoptotic marker XIAP (p = 0.0013). DLBCL cases with nuclear expression of NF-κB showed a significantly poorer overall survival as compared to those without NF-κB expression (p = 0.0236). In a multivariate analysis using a Cox proportional hazard model for IPI and NF-κB expression, the relative risk was 2.97 for high NF-κB expression (95% CI 1.27-6.94; p = 0.0113) and 7.55 for the high-IPI group (95% CI 3.34-18.35; p < 0.0001). In vitro, Bay 11-7085 inhibited constitutively active NF-κB expression in a dose-dependent manner and inhibition of NF-κB also down-regulated expression of the downstream target gene products Bcl-2, Bcl-XL (BCL2L1), XIAP and Survivin, leading to apoptosis via activation of the mitochondrial apoptotic pathway. NF-κB over-expression was found to be an independent prognostic marker for poor survival in DLBCL. Altogether, these results suggest that NF-κB may be a useful prognostic biomarker and a potential target for therapeutic intervention in DLBCL.

Low-grade lymphoma: Beyond fludarabine-single therapy

Low-grade lymphoma: Beyond fludarabine-single therapy

A methylene chloride fraction of Saururus chinensis induces apoptosis through the activation of caspase-3 in prostate and breast cancer cells

The aerial parts of Saururus chinensis (SC) have been used for the treatment of edema, fever, jaundice, and inflammatory diseases in Korean folk medicine for centuries. However, the mechanism by which SC exerts these anti-tumorigenic activities in human prostate and breast cancer cells has not yet been fully understood. In this study, we report on the methylene chloride fraction from SC exerting cytotoxicity against prostate and breast cancer cells in a dose-dependent manner. Specifically, SC exerted the most potent cytotoxicity in LNCaP and MCF-7 cells. SC was shown to down-regulate various angiogenetic (VEGF), proliferative (Cyclin D₁, anti-apoptotic (Bcl-2) gene products in these cells. SC also increased the number of annexin V-positive apoptotic bodies and the sub-G1 DNA contents of the cell cycle undergoing apoptosis through caspase-3 activation in both LNCaP and MCF-7 cells. We further confirmed that caspase-3 plays an important role in SC-induced apoptosis in LNCaP and MCF-7 cells through the use of the caspase-3 inhibitor. Moreover, we observed that SC potentiated paclitaxel-induced apoptosis in MCF-7 cells and sauchinone is a major active constituent of SC, which could induce apoptosis in the cells. Taken together, our data provide the evidence that SC induces apoptosis depending on caspase-3 activation and overcomes the natural biological resistance to chemotherapy found in human prostate and breast cancer cells.

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (³30%) negatively influenced the disease- free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl- 2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for Bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.

Targeting the Bcl-2

Members of the Bcl-2 family of proteins are critical components in regulating the intrinsic apoptotic pathway. Bcl-2 protein overexpression is associated with drug resistance and poor clinical outcome in cancer patients. Preclinical and clinical evaluations demonstrate that downregulation of Bcl-2 restores the intrinsic apoptotic pathways with antitumor effects. Thus, Bcl-2 is aggressively pursued as a therapeutic target in cancer with several new drugs undergoing clinical investigations. In this manuscript, we will review clinical information on some of the novel compounds specifically designed to target the Bcl-2 gene product(s). Extensive clinical evaluations using a Bcl-2-specific antisense have resulted in an overall disappointing experience. But new small molecule inhibitors of the Bcl-2 hold promise with high target affinity, ease of administration and improved toxicity profile. Early stage clinical trials of these agents are revealing promising results alone as well as in combination with existing anticancer therapeutics. Encouraging results from some of these clinical investigations are summarized in this review. Downregulation of Bcl-2 and restoration of a critical apoptotic pathway in cancer cells remains an important strategy. Novel Bcl-2 inhibitors have started to deliver the therapeutic promise of this target-specific quest.

Curcumin Modulates the Radiosensitivity of Colorectal Cancer Cells by Suppressing Constitutive and Inducible NF-κB Activity

Radiation therapy is an integral part of the preoperative treatment of rectal cancers. However, only a minority of patients achieve a complete pathologic response to therapy because of resistance of these tumors to radiation therapy. This resistance may be mediated by constitutively active pro-survival signaling pathways or by inducible/acquired mechanisms in response to radiation therapy. Simultaneous inhibition of these pathways can sensitize these tumors to radiation therapy. Human colorectal cancer cells were exposed to clinically relevant doses of gamma rays, and the mechanism of their radioresistance was investigated. We characterized the transcription factor nuclear factor-kappaB (NF-kappaB) activation as a mechanism of inducible radioresistance in colorectal cancer and used curcumin, the active ingredient in the yellow spice turmeric, to overcome this resistance. Curcumin inhibited the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation stimulated NF-kappaB activity in a dose- and time-dependent manner, whereas curcumin suppressed this radiation-induced NF-kappaB activation via inhibition of radiation-induced phosphorylation and degradation of inhibitor of kappaB alpha, inhibition of inhibitor of kappaB kinase activity, and inhibition of Akt phosphorylation. Curcumin also suppressed NF-kappaB-regulated gene products (Bcl-2, Bcl-x(L), inhibitor of apoptosis protein-2, cyclooxygenase-2, and cyclin D1). Our results suggest that transient inducible NF-kappaB activation provides a prosurvival response to radiation that may account for development of radioresistance. Curcumin blocks this signaling pathway and potentiates the antitumor effects of radiation therapy.

Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic pathway in leukemic cells

Methyl angolensate (MA), a natural tetranortriterpenoid, purified from Soymida febrifuga is examined for the first time for its anticancer properties. We find that MA inhibits growth of T-cell leukemia and chronic myelogenous leukemia cells in a time- and dose-dependent manner. Accumulation of cells in the subG1 peak, annexin V binding and DNA fragmentation suggested induction of apoptosis. Besides, upregulation of BAD (proapoptotic) and downregulation of BCL2 (antiapoptotic) gene products further supported induction of apoptosis. Loss of mitochondrial membrane potential, activation of caspase 9, caspase 3, cleavage of PARP, downregulation of Ku70/80 and phosphorylation of MAP kinases suggested that MA could induce intrinsic pathway of apoptosis in leukemic cells.

Acute hydrodynamic forces and apoptosis: A complex question

A second generation flow contraction device was developed and modeled which allows cells to be subjected to well-defined hydrodynamic forces. Studies were conducted with this system on wild-type Chinese Hamster Ovary cells (CHO-K1) and a strain of CHO cells which expresses the human Bcl-2 triangle gene (CHO-bcl-2). In this study, the following questions were asked: (1) Does an acute hydrodynamic force induce apoptosis in wild-type CHO and CHO-bcl-2 cells? (2) Does the type of culture media make a difference with respect to the induction of apoptosis or necrosis? and (3) Does culture history affect induction of apoptosis or necrosis? The results obtained with this new flow contraction device and corresponding computer simulations are consistent with previously published studies with respect to the level of energy dissipation rate (EDR) required to create significant cell lysis. Second, while detectable relative to the control in the T-flask experiments, only a small fraction of the cells become apoptotic when exposed to a sub-lysis level of EDR (<10(8) W x m(-3)). Third, cells cultured in suspension with serum free media do not exhibit any higher or lower sensitivity (with respect to apoptosis) to various levels of EDR when compared to control cultures grown in T-flask and serum containing media; on the other hand, necrosis is significantly increased in experiments performed on suspended cells without serum. Fourth, the addition of the Bcl-2 gene product might slightly reduce the occurrence of apoptosis in T-flask culture; however, the baseline response is so low that the difference is insignificant.