Rajendra G. Mehta

Expression of Vitamin D Receptor and 25-Hydroxyvitamin D3-1α-Hydroxylase in Normal and Malignant Human Colon

Considerable evidence exists to support the use of vitamin D to prevent and/or treat colorectal cancer. However, the routine use of bioactive vitamin D, 1,25-dihydroxyvitamin D3, is limited by the side effect of toxic hypercalcemia. Recent studies, however, suggest that colonic epithelial cells express 25-hydroxyvitamin D3-1α-hydroxylase, an enzyme that converts nontoxic pro-vitamin D, 25-hydroxycholecalciferol [25(OH)D3], to its bioactive form. Yet, nothing is known as to the cellular expression of 1α-hydroxylase and the vitamin D receptor (VDR) in the earliest histopathologic structures associated with malignant transformation such as aberrant crypt foci (ACF) and polyps [addressing the possibility of using nontoxic 25(OH)D3 for chemoprevention], nor is anything known as to the expression of these proteins in colorectal cancer as a function of tumor cell differentiation or metastasis [relevant to using 25(OH)D3 for chemotherapy]. In this study, we show that 1α-hydroxylase is present at equal high levels in normal colonic epithelium as in ACFs, polyps, and colorectal cancer irrespective of tumor cell differentiation. In contrast, VDR levels were low in normal colonic epithelial cells; were increased in ACFs, polyps, and well-differentiated tumor cells; and then declined as a function of tumor cell de-differentiation. Both 1α-hydroxylase and VDR levels were negligible in tumor cells metastasizing to regional lymph nodes. Overall, these data support using 25(OH)D3 for colorectal cancer chemoprevention but suggest that pro-vitamin D is less likely to be useful for colorectal cancer chemotherapy.

Functional Significance of Vitamin D Receptor FokI Polymorphism in Human Breast Cancer Cells

Background The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)2D3, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)2D3 downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined. Methods/Findings To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)2D3 treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively. Conclusions/Significance These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.

Paxillin expression and amplification in early lung lesions of high-risk patients, lung adenocarcinoma and metastatic disease

Background Paxillin is a modular protein that localises to cell adhesion sites where it facilitates bidirectional communication between the intracellular actin cytoskeleton and the extracellular matrix. These complex and dynamic interactions are essential for cell adhesion, cell migration and cell survival. The authors have previously demonstrated that paxillin is overexpressed in lung cancer tissues and identified somatic paxillin mutations in 9% of lung cancers. A murine in vivo xenograft model of the most common paxillin mutation (A127T) showed increased cell proliferation and invasive tumour growth, establishing an important role for paxillin in the development of lung cancer. Methods The authors analysed 279 bronchoscopy-aided biopsy specimens from 92 high-risk patients. Adenocarcinoma with bronchioloalveolar features and pure bronchioloalveolar carcinoma (BAC) were analysed with fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC). Results Paxillin is overexpressed in premalignant areas of hyperplasia, squamous metaplasia and goblet cell metaplasia, as well as dysplastic lesions and carcinoma in high-risk patients. Concordance between increased paxillin gene copy number and paxillin overexpression was observed in cases of adenocarcinoma eusomic for chromosome 12. Conclusions Paxillin overexpression occurs during the earliest stages of lung cancer development. FISH and IHC analysis of lung adenocarcinoma suggests that relatively small-scale genomic rearrangements of chromosome 12 are associated with paxillin overexpression in lung adenocarcinoma.

Anticarcinogenic Effects of Retinoids in Animals

Retinoids are effective inhibitors of chemical carcinogenesis in the skin, mammary gland, esophagus, respiratory tract, pancreas, and urinary bladder of experimental animals. Modification of the basic retinoid structure has produced retinoids with enhanced target organ specificity, resulting in increased anticancer activity with reduced systemic toxicity. Newer retinoidal benzoic acid derivatives are even more active. Combining retinoid treatment with other modulators of carcinogenesis results in a synergistic inhibition of tumor development. Retinoids in combination with hormonal manipulation are much more effective in inhibiting mammary carcinogenesis than is either treatment alone; this combination approach also inhibits mammary tumor recurrence following surgical removal of the first tumor. Retinoids are most effective when administered shortly after the carcinogenic insult. However, even when retinoid treatment is delayed, the compounds are still effective cancer chemopreventive agents for the mammary gland and urinary bladder. The time that retinoid exposure can be delayed and retain an anticancer effect is directly related to tumor latency, with a longer delay permissible against tumors with long latent periods. The mechanism(s) by which retinoids inhibit carcinogenesis is unknown; however, in the mammary gland, retinoids inhibit differentiation and proliferation, DNA synthesis, and RNA polymerase activity. Cytosolic retinoid-retinoid receptor complexing is apparently a prerequisite for the nuclear interaction of retinoids, at least in mammary cells.

Androgen Receptor regulation of Vitamin D receptor in response of castration-resistant prostate cancer cells to 1α-Hydroxyvitamin D5 - a calcitriol analog.

Calcitriol (1,25(OH)(2)D3) is cytostatic for prostate cancer (CaP), but had limited therapeutic utility due to hypercalcemia-related toxicities, leading to the development of low-calcemic calcitriol analogs. We show that one analog, 1-α-Hydroxyvitamin-D5 (1α(OH)D5), induced apoptosis in castration-sensitive LNCaP prostate cancer cells, but unlike calcitriol, did not increase androgen receptor (AR) transcriptional activity. LNCaP-AI, a castrate-resistant (CRCaP) LNCaP subline, was resistant to 1α(OH)D5 in the presence of androgens; however, androgen withdrawal (AWD), although ineffective by itself, sensitized LNCaP-AI cells to 1α(OH)D5. Investigation of the mechanism revealed that the vitamin D receptor (VDR), which mediates the effects of 1α(OH)D5, is downregulated in LNCaP-AI cells compared to LNCaP in the presence of androgens, whereas AWD restored VDR expression. Since LNCaP-AI cells expressed higher AR compared to LNCaP and AWD decreased AR, this indicated an inverse relationship between VDR and AR. Further, AR stimulation (by increased androgen) suppressed VDR, while AR downregulation (by ARsiRNA) stimulated VDR levels and sensitized LNCaP-AI cells to 1α(OH)D5 similar to AWD. Another cell line, pRNS-1-1, although isolated from a normal prostate, had lost AR expression in culture and adapted to androgen-independent growth. These cells expressed the VDR and were sensitive to 1α(OH)D5, but restoration of AR expression suppressed VDR levels and induced resistance to 1α(OH)D5 treatment. Taken together, these results demonstrate negative regulation of VDR by AR in CRCaP cells. This effect is likely mediated by prohibitin (PHB), which was inhibited by AR transcriptional activity and stimulated VDR in CRCaP, but not castrate-sensitive cells. Therefore, in castration sensitive cells, although the AR negatively regulates PHB, this does not affect VDR expression, whereas in CRCaP cells, negative regulation of PHB by the AR results in concomitant negative regulation of the VDR by the AR. These data demonstrate a novel mechanism by which 1α(OH)D5 prolong the effectiveness of AWD in CaP cells.

Suppression of Breast Cancer Cell Proliferation by Selective Single-Domain Antibody for Intracellular STAT3:

Background: The serendipitous discovery of heavy-chain antibodies devoid of light chains in camelids and the subsequent development of VHHs (variable region of camelid heavy chain) have provided a very important tool for research and possibly for therapeutics. In this study, we synthesized single-domain 15-kDa antibody SBT-100 (anti-STAT3 B VHH13) against human STAT3 (signal transducer and activator of transcription) that binds selectively to STAT3 and suppresses the function of phosphorylated STAT3 (p-STAT3). Methods: Single-chain VHH nanobodies were generated by immunizing camelid with humanized STAT3. Commercially available breast cancer cell lines including MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, and BT474 were used. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The association of anti-STAT3 B VHH13 with STAT3 and p-STAT3 was determined by immunoprecipitation and Western blot analyses. The efficacy of SBT-100 on the growth of MDA-MB-231 xenografts in vivo was determined using athymic mice. Statistical significance for cell proliferation was determined using analysis of variance. If a significant difference (Pu2009<u2009.05) was observed, then Tukey-Kramer multiple comparison test was conducted. Results: SBT-100 suppressed cell proliferation of triple-negative breast cancer cells (Pu2009<u2009.01) as well as provided significant inhibition of tumor growth (Pu2009<u2009.05) in a xenograft model without any toxicity. Results are presented to show that anti-STAT3 B VHH13 selectively binds to STAT3 suggesting that the effects were mediated by inhibiting STAT3. Conclusions: A very large number of human malignancies and benign diseases have constitutive STAT3 activation. Therefore, the results described here suggest that anti-STAT3 B VHH13 can be developed for therapeutic intervention for cancer cells expressing STAT3 or p-STAT3.

Enhanced detection of early photons in time-domain optical tomography using dead-time characteristics of SPADs

A novel approach is presented for enhancing the detection of “early” photons in time-domain optical tomography utilizing the dead time effect of the detector to shift the dynamic range of it towards the first arrival photons.