Apoptosis is a form of programmed cell death characterized by nuclear chromatin condensation, cell shrinkage, membrane blebbing, and DNA fragmentation. [1][2][3] The relationship between intracellular acidification and apoptosis has been studied. [4][5][6][7][8][9] In recent studies, the intracellular acidification may lead to activation of endonucleases and induce apoptosis in tumor cells. 6,8,9 In addition, the role of intracellular pH (pHi) in apoptosis and cell proliferation has been investigated. 10,11 The pHi of cancer cells has been reported to be more alkaline than that of normal cells, and the maintenance of a neutral or slightly alkaline pHi is required for cell growth, transformation, and metabolism. 10,11 Therefore, increasing the intracellular acidity by inhibiting the pHi regulatory mechanisms is cytotoxic and suggests that the pHi regulatory mechanisms may serve as targets for tumor therapy. 6,8,9 Prodigiosins (prodigiosin, metacycloprodigiosin, and prodigiosin 25-C) are red pigments produced as chromophores by various bacteria including Serratia marcescens, Pseuodomonas magnesiorubera, and others. 12 Among the prodigiosin family, prodigiosin 25-C inhibited H ϩ translocation by vacuolar-type H ϩ -ATPase (V-ATPase) without any effect on its ATP hydrolytic activity and suppressed the growth of neoplastic Chinese hamster ovary cells. 13 We previously reported that cycloprodigiosin hydrochloride (cPrG-HCl), which is a member of the prodigiosin family and is more pure and stable than the others, inhibited H ϩ translocation by V-ATPase in the same manner as other prodigiosins. 14 Recently, it was found that prodigiosins promote H ϩ /Cl Ϫ symport across vesicular membranes, resulting in an uncoupling of V-ATPase. 13,15,16 Therefore, these reports suggested that prodigiosins are useful pH regulators and may be promising anticancer drugs.To date, the nature of the interaction between the alteration of pHi and apoptosis induced by cPrG-HCl has not been investigated. Therefore, in this study, we have shown that cPrG-HCl suppressed the cellular proliferation and induced apoptosis as a result of a decrease of pHi on liver cancer cell lines. MATERIALS AND METHODSCell Culture. Six liver cancer cell lines (Huh-7, HCC-M, and HCC-T, human hepatocellular carcinoma; HepG2, human hepatoAbbreviations: pHi, intracellular pH; V-ATPase, vacuolar-type H ϩ -ATPase; cPrG-HCl, cycloprodigiosin hydrochloride; DMEM, Dulbecco' s modified Eagle minimum essential medium; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-2-yl), 5 diphenyltetrazolium bromide; IC 50 , 50% inhibitory concentration; BCECF-AM, 2Ј,7Ј-bis-(Carboxyethyl)-5(6Ј)-carboxyfluorescein acetoxymethyl ester; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling.From the

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SummaryMany colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-β (TGF-β). TGF-β also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-β and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-β 1 , type I and type II receptor mRNA and immunohistochemical staining of TGF-β 1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-β receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-β receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-β 1 mRNA and the protein were detected in all colorectal cancers. TGF-β receptor mRNAs and TGF-β 1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-β stimulation. The escape of human colon cancer from TGF-β -mediated growth inhibition by down-regulation of TGF-β receptors as well as the effects of TGF-β on stroma formation and angiogenesis indicate a possible role for TGF-β in the progression of colon cancer in an intact host.Keywords: TGF-β; TGF-β receptor; Smad, colorectal cancer; colorectal adenoma 194British Journal of Cancer (1999) 80(1/2), 194-205 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 30 receptors, and alterations in post-receptor pathways. Several reports indicate that elevated levels of TGF-β mRNA and protein in colorectal cancer are associated with cancer progression (Tsushima et al, 1996). If TGF-β is indeed an inhibitor for epithelial cells, how then do colorectal cancer cells manage to proliferate despite elevated TGF-β production by tumour cells? Concerning the mechanisms of resistance to the anti-proliferative effects of TGF-β in colorectal cancer, Markowitz et al (1995) identified a specific TGF-β RII mutation that is associated with defective DNA mismatch repair in colon cancer cells. However, numerous reports also indicate that transcriptional regulation makes an important contribution to determination of the expression level of TGF-β receptors (Birchenall-Roberts et al, 1995;Kim et al, 1997). Accordingly, to clarify the mechanisms of resistance to the antiproliferative effects of TGF-β in vivo, we estimated the transcriptional level of TGF-β receptors in colorectal cancer using in situ hybridization and Northern blot hybridization as the standard for those in normal colorectal epithelial cells.In addition, no studies of human colorectal tumour to date have compared in situ levels of TGF-β 1 with those of its receptors as a presumptive target gene, and with the presence of ligand protein.To ascertain if TGF-β from colorectal cancer act...

The effect of cycloprodigiosin hydrochloride (cPrG.HCl), a H+/Cl- symporter, on five human breast cancer cell lines (KPL-1, T-47D, MCF-7, MKL-F, and MDA-MB-231), a human breast epithelial cell line (HBL-100), and a human fibroblast cell line (WI-38-40) was examined. cPrG.HCl inhibited the growth of all five breast cancer cell lines (IC50: 0.46-0.62 microM) and slightly inhibited HBL-100 and WI-38-40 cell growth (IC50: 1.75 microM and 2.26 microM respectively). cPrG.HCl treatment in KPL-1 cells increased the pH of acidic organelles, decreased intracellular pH, and caused apoptosis, which was confirmed by the appearance of a sub-G1 population by flow cytometry and DNA fragmentation. In addition, cPrG.HCl-induced apoptosis was strongly suppressed by imidazole, a cell-permeable base, suggesting that intracellular acidification was essential for the apoptosis. Further, cPrG.HCl treatment up-regulated Bax and Bak expression, down-regulated Bcl-2 expression, and activated caspase-3. Therefore, the intracellular acidification by cPrG.HCl treatment suppressed the growth of human breast cancer cell lines by inducing apoptosis.

Cycloprodigiosin hydrochloride (cPrG • HCl), a novel H+/Cl– symporter, induces acidification of the cytosol and leads to apoptosis in rat and human liver cancer cells. In the present study, the effect of cPrG • HCl on a promyelocytic leukemia cell line (HL‐60) was examined. cPrG • HCl lowered intracellular pH and induced apoptosis through up‐regulation of Fas ligand, activation of stress‐activated protein kinase (SAPK/JNK) and caspase. Apoptosis induced by cPrG • HCl was strongly suppressed when a cell‐permeable weak base, imidazole, was present, indicating that cytosol acidification introduced by cPrG • HCl triggered caspase activation, leading to apoptosis. Concomitantly, cell differentiation into monocyte was also induced by cPrG • HCl both morphologically and functionally. However, the cPrG • HCl‐induced differentiation was not suppressed by addition of imidazole, indicating that the differentiation process is unrelated to cytosol acidification. Further, the differentiation induced by cPrG • HCl was blocked by tyrosine kinase inhibitors (lavendustin A and HMA) but unaffected by the inhibitors of A‐kinase (H‐89) or C‐kinase (H‐7). Taken together, these findings suggest that cPrG • HCl, through apoptosis and differentiation induction, may be useful in leukemia treatment. Int. J. Cancer 88:121–128, 2000. © 2000 Wiley‐Liss, Inc.