Expression of gamma-aminobutyric acid receptor (subtype A) in prostate cancer.

BACKGROUND: In prostate cancer, gamma-aminobutyric acid (GABA) has been previously reported to increase cellular proliferation via the ionotropic GABAa receptor (GABAar) and to promote cellular invasiveness via the metabotropic GABAb receptor. METHODS: In this study, we have investigated, by immunohistochemistry, GABAar levels in 12 normal human prostate, 13 benign prostatic hyperplasia (BPH) and 148 human prostate cancer specimens. We have also examined the effect of several GABA agonists and antagonists on the in vitro proliferation of four human prostate cancer cell lines: LNCaP, MDA-PCA-2b, DU145 and PC3. RESULTS: GABAar immunoreactivity was present in the stroma of ~75% of the normal and BPH specimens, and in 95% of the prostate cancer specimens. Also, low to moderate GABAar staining was observed in the acinar epithelium of 50 (33%) prostate cancer specimens. No correlation was observed between GABAar staining and patient age, Gleason Sum or TNM stage. A GABAa agonist isoguvacine, at doses between 5-50 microg/ml (31-310 microM), stimulated the proliferation of all four human prostate cancer cell lines, tested. Baclofen, a GABAb agonist (up to 50 microg/ml, 234 microM) had no effect on growth. Also, at concentrations up to 100 microg/ml, GABA antagonists, bicuculline (223 microM), picrotoxin (166 microM) and saclofen (400 microM), did not have significant growth-inhibitory effects. However, dihydroergotoxine, which binds the GABAar chloride ion-channel, inhibited cellular proliferation (IC(50) 18-38 microM). CONCLUSIONS: These data indicate frequent expression of GABAar in prostate cancer and support a role for GABAar in the proliferation of prostate cancer cells.

Ryanodine receptor expression correlates with tumor grade in breast cancer.

Ryanodine receptors (RyRs) have been previously implicated in the proliferation of human T-lymphocytes and melanocytes as well as in the motility of astrocytes. We have examined RyR expression in 57 ductal, human breast cancer specimens, by immunohistochemistry. Moderate to high RyR immunostaining was found in 47 (82%) of the specimens. There was a direct correlation between RyR levels and tumor grade (r = 0.48, p = 0.0002). We have also examined the effect of the RyR agonist 4-chloro-m-cresol on the in-vitro growth of two human breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with 4-chloro-m-cresol inhibited the growth of both breast cancer cell lines, in a dose-dependent manner, with half-maximal inhibition observed at 30 to 50 microg/mL (210-351 microM). Our data suggest that RyR could serve as prognostic indicator and/or as a target for breast cancer treatment.

Reduced Kv1.3 potassium channel expression in human prostate cancer.

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = -0.25, P = 0.003) as well as tumor stage (r = -0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.

Potentiation of the antiproliferative activity of MKT-077 by loperamide, diltiazem and tamoxifen.

MKT-077, a delocalized lipophilic cation, selectively targets cancer cells. MKT-077 has been reported to inhibit the growth of several tumor types and has undergone phase I clinical testing. We have examined the effect of MKT-077, alone and in combination with the antidiarrheal drug loperamide. Ten human cancer cell lines, four prostate (PC3, DU145, LNCaP, MDA-PCA-2B), two breast (MCF-7 and MDA-MB-231) and four colon (LoVo, Colo320DM, SW1116 and LS174t) were tested in vitro. Cells were grown to confluency prior to treatment. Loperamide potentiated the antiproliferative effect of MKT-077 in all ten cell lines, in a dose-dependent manner. The sensitivity of MDA-PCA-2B cells, the two breast and four colon cancer cell lines to MKT-077 was relatively low (>2.5 microg/ml MKT-077 required to inhibit growth by 95%). In these cell lines, 0.5-5 microg/ml (1-10 microM) loperamide caused a marked increase in the response to MKT-077. Loperamide is known to activate micro-opioid receptors at nanomolar concentrations and block voltage-gated calcium channels at micromolar doses. We found that calcium channel-blockers diltiazem and nifedipine (10-20 microg/ml), as well as tamoxifen (1.5-2.5 microg/ml) can also potentiate the growth-inhibitory effects of MKT-077. These synergistic interactions could be exploited for therapeutic benefit.

Activity of potassium channel-blockers in breast cancer.

BACKGROUND: Potassium ion (K+) channels are known to play a key role in breast cancer proliferation. MATERIALS AND METHODS: We investigated the expression of Kv1.3 voltage-gated K+ channels in 60 human breast cancer specimens by immunohistochemistry. The effects of K+ channel-blockers on cellular proliferation were examined in vitro. RESULTS: No immunostaining was observed in 4 normal human breast specimens. Eighteen (30%) breast cancer specimens showed high, 35 (58%) moderate and 7 (12%) low Kv1.3 staining in the epithelial compartment. Minoxidil (K+ channel-opener) stimulated growth of MCF-7 human breast cancer cells (maximal approximately 60% at 10 micrograms/mL). K+ channel-blockers, dequalinium and amiodarone, had marked inhibitory effects on MCF-7 proliferation (> 90% inhibition at 1.5 micrograms/mL). Importantly, amiodarone and dequalinium potentiated the growth-inhibitory effects of tamoxifen on human breast (MCF-7, MDA-MB-231) as well as prostate (PC3, MDA-PCA-2B) and colon (Colo320DM, SW1116) cancer cell lines. CONCLUSION: Investigation of combination therapy with tamoxifen and K+ channel-blockers is warranted.

An assessment of the in vitro antimicrobial effects of two antiepileptic drugs--sodium valproate and phenytoin.

An incidental observation led to the evaluation of the antimicrobial properties of valproate and phenytoin. In vitro inhibition of Mycobacterium smegmatis, Candida albicans, and other standard test organisms by these two antiepileptic drugs was assessed using the broth microdilution procedure. Fluorescence microscopy (Viability Stains, Molecular Probes Inc.) and cultural techniques were employed to distinguish between microbicidal and microbistatic effects. Phenytoin showed no inhibitory activity against the microbes tested. Sodium valproate, on the other hand, was selectively potent against the yeast strains in a dose-dependent manner (MIC = 10-20 microg ml(-1)). In vitro activity against Mycobacterium smegmatis (70% growth inhibition by 81 microg ml(-1)) was moderate to low while Staphylococcus aureus. Pseudomonas aeruginosa and Escherichia coli were not significantly affected. Viability data from fluorescent microscopy and plate cultures correlated well with absorbance (A620nm) growth index, and showed that valproate was microbicidal against susceptible organisms. The mode of action may include blockage of calcium channels and perturbation of membrane potential. This report opens up yet another opportunity for further enquiry into the fundamental mechanisms of drug action and microbial resistance.

Expression and activity of potassium ion channels in human prostate cancer.

Four normal and 79 human prostate cancer (Pca) specimens were examined, by immunohistochemistry, for expression of voltage-gated potassium ion channels. Strong immunostaining (for Kv1.3) was observed in the normal and 47% (37/79) of Pca specimens. Twenty-nine percent (23/79) Pca specimens showed moderate and 24% (19/79) displayed low staining. Three potassium channel-openers at a concentration of 10 microg/mL, minoxidil (47.8 microM), 1-Ethyl-2-benzimidazolinone (EBIO) (61.7 microM) and diazoxide (43.3 microM), increased growth of PC3 cells by 30-50%. Potassium channel-blockers, dequalinium, amiodarone and glibenclamide, caused a dose-dependent, growth inhibition of four human Pca cell lines. Apoptosis occurred within 4h of treatment of PC3 cells with dequalinium (0.5 microg/mL, 0.9 microM), amiodarone (5 microg/mL, 7.3 microM) or glibenclamide (50 microg/mL, 0.1mM).

Voltage-gated sodium ion channels in prostate cancer: expression and activity.

BACKGROUND: High levels of voltage-gated sodium channels (VGSCs) have been previously associated with the invasiveness of rat and human prostate cancer (Pca) cell lines. MATERIALS AND METHODS: 4 normal prostate and 80 clinical Pca specimens on a tissue microarray slide were examined by immunohistochemistry using anti-sodium channel (III-IV linker region) antibody. The effects of a VGSC-opener and VGSC-blockers on the in vitro proliferation of 4 human Pca cell lines was examined. RESULTS: Fifty-five% (44 out of 80) Pca specimens showed higher VGSC levels compared to normal, with 14 (of 44) showing increased focal staining. VGSC-opener veratrine (1-50 microg/mL), increased growth of PC3, DU145, LNCaP and MDA-PCA-2B Pca cells. VGSC-blockers, flunarizine (IC50=2 microg/mL) and riluzole (IC50=10-30 microg/mL) caused dose-dependent growth-inhibition of all four cell lines. Western analysis of cell extracts showed VGSC-immunoreactivity in the 4 Pca cell lines. CONCLUSION: Our results indicate increased expression of VGSCs in Pca and VGSC involvement in Pca growth.

Voltage-gated potassium ion channels in colon cancer.

Voltage-gated potassium channels (VGPCs) have been previously implicated in cellular proliferation. In this study, the expression of VGPCs was examined by immunohistochemistry in seventy-four human colonic carcinoma specimens. Immunostaining for the Kv1.3 type VGPC was absent in two normal human colon specimens. Kv1.3 staining in the 74 colon cancer specimens was low in 9% (7/74), moderate in 61% (45/74) and high in 30% (22/74). Potassium channel (PC) openers, minoxidil and diazoxide (5-50 microg/ml), increased growth of SW1116, LoVo, Colo320DM and LS174t human colon cancer cell lines by 20-40%. PC-blockers, dequalinium and amiodarone, caused marked growth-inhibition of the four cell lines, at concentrations between 1 to 3 microg/ml. PC-blockers such as glibenclamide inhibited cellular proliferation at concentrations above 50 microg/ml while tetraethylammonium and 4-aminopyridine (up to 100 microg/ml) did not have significant growth-suppressive effects. Our results indicate the presence of VGPCs in colon cancer and suggest that PCs could serve as therapeutic targets.

Relationship of the interleukin-1 system with neuroendocrine and exocrine markers in human colon cancer cell lines.

Interleukin 1 (IL-1) is known to regulate the proliferation and differentiation of normal and malignant immune cells as well as other cell types. Expression of IL-1 (alpha and beta), IL-1 receptors (RI and RII) and IL-1 receptor antagonist (IL-1RA) was determined by RT-PCR in seven human colon carcinoma cell lines (COLO 320DM, LoVo, SW403, SW1116, SW1417, LS123 and LS174t). Influence of IL-1 on the secretion of the neuroendocrine (NE) differentiation marker chromogranin A (CGA) and the exocrine marker carcinoembryonic antigen (CEA) was examined by Western blotting. Our data indicate that CGA and IL-1RI are expressed by all seven, IL-1 beta by five, IL-1RII and IL-1RA by six lines. IL-1 alpha transcripts were found only in three lines (LoVo, SW1116 and LS174t) and correlated with high CEA levels and aggressive growth behavior. "Pure" NE cell lines (COLO 320DM and LS123) secreted the highest levels of CGA, but the lowest levels of CEA and were IL-1 (alpha and beta) negative. Exogenously added IL-1 caused a decrease in CGA, but an increase in CEA secretion. Our results suggest an inverse relationship between IL-1 and NE differentiation, as well as a direct relationship between IL-1 and CEA expression in colon carcinoma.