Madan Lal Brahma Bhatt

Circadian variation in radiation-induced intestinal mucositis in patients with cervical carcinoma†

Mucositis, a radiotherapy‐associated toxicity, is an important factor determining morbidity and treatment compliance. Gastrointestinal mucositis in patients undergoing radiotherapy may also depend on time of administration of radiation in addition to several other factors. The presence of any correlation between the severity of acute gastrointestinal mucositis in cervical carcinoma patients and the time of irradiation was prospectively evaluated.

2‐D gel electrophoresis‐based proteomic analysis reveals that ormeloxifen induces G0–G1 growth arrest and ERK‐mediated apoptosis in chronic myeloid leukemia cells K562

Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose‐dependent manner in a variety of leukemia cells, more strikingly in K562. 2‐DE‐gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell‐cycle pathways, respectively. Our data demonstrate that ormeloxifen‐induced apoptosis in K562 cells involves activation of extracellular signal‐regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria‐mediated caspase‐3 activation. Ormeloxifen‐induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0–G1 phase by inhibiting c‐myc promoter via ormeloxifen‐induced MBP‐1 (c‐myc promoter‐binding protein) and upregulation of p21 expression. We further show that ormeloxifen‐induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0–G1 phase by reciprocal regulation of p21 and c‐myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility.

Role of recombinant human erythropoietin in patients of advanced cervical cancer treated "by chemoradiotherapy".

Background: Cervical cancer, in women, is the second most common cancer world wide, next to breast cancer. During the treatment of carcinoma cervix, anemia is selectively frequent and its origin is complex combining hemorrhage, iron deprivation, inflammatory reactions and infection. The objective of this study is to evaluate the role of epoetin in correction of anemia and on treatment outcomes in patients with advanced cervical cancer receiving concurrent chemoradiotherapy. Results: A total of 120 patients were enrolled in the study of which 60 patients were randomized to receive epoetin beta in the treatment arm and 60 patients were in control arm where epoetin beta was not given. Total two and three patients absconded during treatment from treatment and control arm respectively; therefore total evaluable patients were 115. Mean Hb at baseline in the control arm was 10.70 +/- 0.62 g/dl and 10.45 +/- 0.43 g/dl in the treatment arm (p = NS). At the end of treatment mean Hb increased by 1.55 g/dl in patients receiving epoetin beta (p < 0.01), but decreased by 1.50 g/dl in the control arm (p < 0.01). There was significant reduction in blood transfusion in patients receiving epoetin beta (p < 0.01). At the end of treatment there was also significant improvement in energy level, activity level and overall quality of life in the treatment arm (p < 0.01). There was no significant difference in overall survival (p > 0.05), or disease free survival (p > 0.05) between the two study arms. Adverse events were well matched between the two study arms. No Thromboembolic events associated with epoetin beta was observed in our study. Material and methods: Total 120, stage IIB to IIIB cervical cancer patients, aged 18-70 years with 9.50-12.50 g/dl baseline Hb value who were to receive radiotherapy together with cisplatin were randomized to receive either epoetin beta 10,000 IU thrice weekly and oral iron starting 10-15 days before their 5-week course of whole pelvic irradiation and weekly cisplatin (treatment arm) or standard supportive care (control arm), where epoetin beta was not given. Blood transfusion was given in patients of both the arms if hemoglobin was =10 g/dl. Conclusions: Treatment with epoetin beta safely and effectively corrects anemia in patients with advanced cervical cancer receiving chemoradiotherapy and is not associated with adverse effects on response rate, overall survival, disease free survival and chemoradiotherapy related acute and late toxicities.

Expression of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) in human urinary bladder transitional cell carcinoma.

The objective of this study was to evaluate the expression pattern of PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) and its clinical significance in human bladder cancer (BC). We detected PBK/TOPK mRNA overexpression in BC and human normal testis tissues using RT-PCR. Using qRT-PCR revealed a higher expression of PBK/TOPK in BC tissues than their adjacent noncancerous tissues (ANCTs) (p<0.0001). Cytoplasmic expression of PBK/TOPK protein was found to be positive in 64.6% (42 of 65) BC patients. Expression of PBK/TOPK protein was found to be significantly higher in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC) (86.1% vs. 37.9%, p<0.001). The immunohistochemical (IHC) expression of PBK/TOPK was found to be significantly (p<0.001) associated with the stage of disease. Study findings suggest that the PBK/TOPK mRNA/protein expression is specific to human BC and might be used as a novel target for development of cancer immunotherapy and diagnostic biomarker.

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells.

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone‐responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam‐mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6‐associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome‐mediated degradation. Furthermore, we show that Tam‐ and siE6AP‐mediated inhibition of E6AP leads to enhanced G0–G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome‐c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam‐targeted E6AP inhibition is in fact required for Tam‐mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.

Expression and clinical significance of Centrosomal protein 55 (CEP55) in human urinary bladder transitional cell carcinoma.

Bladder cancer (BC) is one among the most common and lethal urothelial malignancies worldwide. The expression of cancer-testis (CT) antigens in some tumours and restricted expression among normal tissues make CT antigens as attractive vaccine targets. In this context, we evaluated Centrosomal protein 55 kDa (CEP55), which is specifically expressed in normal human testis and various malignancies. Until the expression pattern of CEP55 in transitional cell carcinoma (TCC) of human urinary bladder and its clinical significance are not known. The aim of the present study is to evaluate mRNA/protein expression of CEP55 in TCCs of urinary bladder and correlate its expression with the clinicopathological characteristics of BC patients. In this study, the methods of quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to investigate mRNA/protein expression of CEP55 in TCC. Independent Students t test, ANOVA and Chi-square (χ(2)) were used to analyze the data statistically. We observed CEP55 mRNA overexpression in testis and 48.7% of BC patients. Relative mean fold expression of CEP55 mRNA was found to be significantly (p<0.01) higher in muscle-invasive bladder cancer (MIBC) as compared to non-muscle-invasive bladder cancer (NMIBC) patients (7.88±3.88 vs. 4.75±2.30, p=0.01). CEP55 protein expression was evaluated using IHC and cytoplasmic staining pattern was recorded in formalin fixed, paraffin-embedded (FFPE) bladder tumour tissues. No significant difference was observed in protein expression of CEP55 between the two groups (NMIBC and MIBC patients) (72.2% vs. 69.0%, p=0.774). No significant protein expression of CEP55 was observed among adjacent noncancerous tissues (ANCTs) and benign prostatic hyperplasia (BPH) used as control. Our study results suggest that CEP55 mRNA/protein expression was observed is specific to TCC of human urinary bladder and might be used as a diagnostic biomarker and vaccine target in development of BC specific immunotherapy.

Expression profiling of G2/M phase regulatory proteins in normal, premalignant and malignant uterine cervix and their correlation with survival of patients

BACKGROUND Cell regulatory G2/M phase proteins are the key regulators of mitosis and have been reported with abnormal expressions in various malignancies. AIM To determine the expressions of these proteins in neoplastic uterine cervix tissue. MATERIALS AND METHODS This study evaluates the G2/M phase regulatory protein expression of Cyclin B1, Aurora-B, Pololike kinase 1 (PLK1) and LIM kinase1 (LIMK1) in tissues of 25 normal (control), 16 dysplastic (dysplasia) and 34 neoplastic (cancer) patients of uterine cervix. The expressions of different proteins were obtained by using Western Blot technique. STATISTICAL ANALYSIS One way analysis of variance (ANOVA), Pearson correlation, Kaplan-Meier and other tests are used for analysis. RESULTS AND CONCLUSION The level of expression of LIMK1 in cervical cancer patients was found to be significantly higher (P < 0.01) than both the controls and dysplasia. The expression of Aurora B and PLK1 in cervical cancer patients was also found to be significantly higher ( P < 0.05) than controls but it did not differ with dysplasia. However, the expression of Cyclin B1 was similar among cervical cancer patients, dysplasia and controls ( P> 0.05). The expression of all the above proteins showed significant ( P < 0.01) and inverse relation with the survival of cancer patients. Among the selected candidate proteins, it was LIMK1 that showed the most positive correlation with the aggressiveness of the disease and negative correlation (r= -0.64; P < 0.01) with the survival of patients.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST‐pull down assay with ERα ligand‐binding domain (ERα‐LBD) and MS‐based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST‐pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα‐mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS‐based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.

A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB

Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low‐abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β‐mercaptoethanol, SDS, and Tris‐HCl and a washing buffer consisting of PBS added with 0.1% Tween‐20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.

Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation of MYO3A gene in breast cancer cells

ABSTRACT The potential of a tumor cell to metastasize profoundly depends on its microenvironment, or “niche” interactions with local components. Tumor-associated-macrophages (TAMs) are the most abundant subpopulation of tumor stroma and represent a key component of tumor microenvironment. The dynamic interaction of cancer cells with neighboring TAMs actively drive cancer progression and metastatic transformation through intercellular signaling networks that need better elucidation. Thus, current study was planned for discerning paracrine communication networks operational between TAMs, and breast cancer cells with special reference to cancer cell invasion and dissemination to distant sites. Here, we report role of MIP-1β in enhancing invasive potential of metastatic breast cancer MDA-MB-231 and MDA-MB-468 cells. In addition, the poorly metastatic MCF-7 cells were also rendered invasive by MIP-1β. The MIP-1β-driven cancer cell invasion was dependent on upregulated expression levels of MYO3A gene, which encodes an unconventional myosin super-family protein harboring a kinase domain. Ex ovo study employing Chick-embryo-model and in vivo Syngenic 4T1/BALB/c mice-model further corroborated aforementioned in vitro findings, thereby substantiating their physiological relevance. Concordantly, human breast cancer specimen exhibited significant association between mRNA expression levels of MIP-1β and MYO3A. Both, MIP-1β and MYO3A exhibited positive correlation with MMP9, an established molecular determinant of cancer cell invasion. Higher expression of these genes correlated with poor survival of breast cancer patients. Collectively, these results point toward so far undisclosed MIP-1β/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1β potentiated cancer cell invasion and metastasis via up regulation of MYO3A gene within cancer cells. Our study exposes opportunities for devising potential anti-metastatic strategies for efficient clinical management of breast cancer.