Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells.

BACKGROUND: Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown. METHODS: In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1. RESULTS: Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)-Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens. CONCLUSION: Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K-Akt signalling pathway and resultant upregulation of cyclin D1 levels.

Hypermethylation-mediated reduction of WWOX expression in intraductal papillary mucinous neoplasms of the pancreas.

We have previously shown that WW domain-containing oxidoreductase (WWOX) has tumour-suppressing effects and that its expression is frequently reduced in pancreatic carcinoma. In this study, we examined WWOX expression in intraductal papillary mucinous neoplasm of the pancreas (IPMN) to assess the function of WWOX in pancreatic duct tumourigenesis using immunohistochemistry and methylation-specific polymerase chain reaction analysis. Among 41 IPMNs including intraductal papillary mucinous adenomas (IPMAs) and intraductal papillary mucinous carcinomas (IPMCs), loss or reduced WWOX immunoreactivity was detected in 3 (15%) of 20 IPMAs and 17 (81%) of 21 IPMCs. In addition, hypermethylation of the WWOX regulatory site was detected in 1 (33%) of 3 WWOX(-) IPMAs and 9 (53%) of 17 WWOX(-) IPMCs, suggesting that hypermethylation may possibly be important in the suppression of WWOX expression. Reduction of WWOX expression was significantly correlated with a higher Ki-67 labelling index but was not correlated with the ssDNA apoptotic body index. Interestingly, decreased WWOX expression was significantly correlated with loss of SMAD4 expression in these IPMNs. The results indicate that downregulation of WWOX expression by the WWOX regulatory region hypermethylation is critical for transformation of pancreatic duct.

Direct cancer-stromal interaction increases fibroblast proliferation and enhances invasive properties of scirrhous-type gastric carcinoma cells.

BACKGROUND: Scirrhous-type gastric carcinoma (SGC) exhibits an extensive submucosal fibrosis and extremely poor patient prognosis. We investigated the importance of the cancer-stromal interaction in the histogenesis of SGC. METHODS: Gastric fibroblasts NF-25 and intestinal fibroblasts NF-j2 were co-cultured with SGC-derived (HSC-39) or non-SGC-derived (HSC-57 and HSC-64) cells. To identify genes that are up- or downregulated in NF-25, complementary DNA (cDNA) microarray analysis was performed. The antibody against vascular-cell adhesion molecule-1 (VCAM-1) was used for cell growth test and immunohistochemistry. Moreover, the impact of interaction with NF-25 fibroblasts on HSC-39 cells was investigated using western blot and reverse transcription-polymerase chain reaction. RESULTS: HSC-39 cells stimulated growth of NF-25 but not NF-j2 when co-cultured. Induction of VCAM-1 in NF-25 fibroblasts was identified, which was specific when co-cultured with HSC-39 but not with non-SGC-derived HSC-57 and HSC-64 cells. Neutralising antibody to VCAM-1 suppressed NF-25 growth in dose-dependent manners. In tissue samples, positive immunoreactivity of VCAM-1 in SGC-derived fibroblasts was significantly higher than that in non-SGC-derived fibroblasts. Furthermore, interaction with NF-25 fibroblasts not only induced the epithelial-mesenchymal transition-like change, but also expressions of matrix metalloproteinase- related genes in HSC-39 cells. CONCLUSION: Direct interaction between SGC cells and gastric fibroblasts establishes the tumour microenvironment and reinforces the aggressiveness of SGC.

High PRL-3 expression in human gastric cancer is a marker of metastasis and grades of malignancies: an in situ hybridization study.

Phosphatase of regenerating liver (PRL)-3, encoding a 22-kD low molecular weight tyrosine phosphatase, has been reported to be associated with metastasis of colorectal carcinoma. We assessed the levels of PRL-3 mRNA expression to know whether its up-regulation was involved in progression and metastasis of gastric carcinoma. Levels of PRL-3 expression in 94 human gastric adenocarcinomas and 54 matched lymph node metastases were detected by in situ hybridization and compared with clinicopathological characteristics including prognosis. High PRL-3 expression was detected in 36.2% of primary gastric carcinoma (with nodal metastasis, 55.6%; without nodal metastasis, 10%; P < 0.001) and in 74.1% of lymph node metastases. The incidence of high PRL-3 expression in lymph node metastasis was significantly higher than in primary tumors (P < 0.044). Moreover, high expression of PRL-3 was closely associated with tumor size, lymphatic invasion, venous invasion, extent of lymph node metastasis, and tumor stage. These results suggest that high PRL-3 expression may participate in the progression and metastasis of gastric carcinoma. PRL-3 might be a novel molecular marker for aggressive gastric cancer.

Cerebral venous thrombosis in adult nephrotic syndrome due to systemic amyloidosis.

Although venous thrombosis is one of the common complications in nephrotic patients, cerebral venous thrombosis (CVT) is rarely reported. CVT is so difficult to be detected by conventional diagnostic methods that it is sometimes overlooked despite its potential severity. We report a 79-year-old female with nephrotic syndrome due to systemic amyloidosis who suddenly altered mental status during her hospitalization. The underlying etiology had been not identified by physical examinations, various laboratory data, and repeated computed tomography, and finally she died. The post-mortem examination showed a massive thrombus impacted in intracranial left-sided transverse and sigmoid sinus. This case suggests that CVT can occur in a nephrotic patient who presents unexplained neurological signs and symptoms, which might not be detected only through conventional diagnostic tests.

Interleukin 1 alpha acts as an autocrine growth stimulator for human gastric carcinoma cells.

The expression and effect of interleukin 1 alpha (IL-1 alpha) were examined in human gastric carcinoma cell lines to determine if IL-1 alpha acts as a growth stimulator for these cells. Six of 8 gastric carcinoma cell lines expressed IL-1 alpha mRNA at various levels. Among them, TMK-1 and MKN-7 cells secreted IL-1 alpha into the culture fluid, in an especially large amount by MKN-7 cells. Scatchard plot analysis of IL-1 alpha binding revealed that TMK-1 cells had only one type of high-affinity receptors, whereas MKN-7 cells had high- and low-affinity receptors. Cell growth and DNA synthesis of TMK-1 and MKN-7 cells were stimulated by IL-1 alpha, and those of MKN-7 were inhibited by addition of anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor, or transforming growth factor alpha. On the other hand, IL-1 alpha increased the mRNA expression for transforming growth factor alpha and epidermal growth factor receptor. These findings indicate that IL-1 alpha is an autocrine growth stimulator for gastric carcinoma cells and the interaction with epidermal growth factor/transforming growth factor alpha/receptor system should be involved in the growth modulation by IL-1 alpha.

An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells.

Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

Growth inhibition of transforming growth factor beta on human gastric carcinoma cells: receptor and postreceptor signaling.

The effect of transforming growth factor beta (TGF-beta) on human gastric carcinoma cell lines was examined. Cell growth and DNA synthesis of TMK-1 were inhibited by TGF-beta, whereas MKN-28 presented no response to TGF-beta. Scatchard plot analysis of TGF-beta binding showed that TMK-1 had a relatively small number of high-affinity receptors, whereas MKN-28 had a large number of low-affinity receptors. By affinity labeling, only the type I receptor (Mr 65,000) for TGF-beta was detected in TMK-1, while three types of receptors, type I, type II (Mr 85,000-95,000), and type III (Mr 250,000-350,000), for TGF-beta were present in MKN-28. TGF-beta treatment reduced p34cdc-2 kinase activity and the level of phosphorylation of retinoblastoma protein in TMK-1, whereas it did not affect them in MKN-28. mRNAs for MYC and platelet-derived growth factor B chain were increased by treatment of TGF-beta on TMK-1. cAMP-responsive element binding activity was decreased by TGF-beta treatment in MKN-28 but not in TMK-1. This was closely correlated with protein kinase C activity. These results suggest that the type I receptor for TGF-beta in human gastric carcinoma cells may be mainly linked with the growth inhibition of TGF-beta by a decrease in retinoblastoma protein phosphorylation by p34cdc-2 without suppression of MYC expression. Conversely, TGF-beta may reduce protein kinase C activity and cAMP-responsive element binding activity in TGF-beta-resistant gastric carcinoma cells.

Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells.

A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.

Inhibition of growth and modulation of gene expression in human lung carcinoma in athymic mice by site-selective 8-Cl-cyclic adenosine monophosphate.

Site-selective cyclic AMP (cAMP) analogues inhibit growth and induce changes in morphology in a spectrum of human cancer cell lines (D. Katsaros et al., FEBS Lett., 223:97, 1987). The cellular events underlying such effects of cAMP analogues include differential regulation of type I versus type II cAMP-dependent protein kinase isozymes (S. Ally et al., Proc. Natl. Acad. Sci. USA, 85: 6319, 1988). Infusion (i.p.) of 8-Cl-cAMP, the most potent site-selective cAMP analogue, for 7 days produced regression of LX-1 lung carcinoma in athymic mice in a dose-dependent manner. The tumor regression correlated with the changing levels of cAMP receptor proteins, RI alpha and RII beta, the regulatory subunits of cAMP-dependent protein kinase type I and type II, respectively. By photoaffinity labeling with 8-N3-[32P]cAMP and immunoblotting with a monospecific anti-RII antibody, RI alpha (Mr 49,000) and RII beta (Mr 51,000) were identified in the untreated control tumors. 8-Cl-cAMP treatment induced a rapid increase of both RI alpha and RII beta in tumor cytosols and translocation (within 1 h) of only RII beta from the cytosol to the nucleus. RII beta in both cytosols and nuclei remained elevated during 8-Cl-cAMP treatment, whereas RI alpha in the cytosols gradually decreased with time of treatment after its initial transient increase. Northern blot analyses demonstrated that the RII beta mRNA level increased within 6 h of 8-Cl-cAMP treatment and remained elevated during treatment, whereas the RI alpha mRNA level decreased to below that of the untreated control tumor level after its transient increase during 1-6 h of treatment. 8-Cl-cAMP treatment also caused a sharp decrease in both N-ras and c-myc mRNA levels. These results suggest that the fundamental basis for the antineoplastic activity of 8-Cl-cAMP may reside in the restoration of normal gene regulation in neoplasms in which cAMP receptor proteins play a role.

Efficient induction of focus formation in a subclone of NIH3T3 cells by c-myc and its inhibition by serum and by growth factors.

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested.

Role of site-selective cAMP analogs in the control and reversal of malignancy.

Two isoforms of cAMP receptor protein, RI and RII, the regulatory subunits of cAMP-dependent protein kinase, transduce opposite signals, the RI being stimulatory and the RII being inhibitory of cell proliferation. In normal cells RI and RII exist at a specific physiological ratio whereas in cancer cells such physiological balance of these receptor proteins is disrupted. Reversal and suppression of malignancy can be achieved when the physiologic ratio of these intracellular signal transducers of cAMP is restored as shown by the use of site-selective cAMP analogs, antisense oligodeoxynucleotides or gene transfer, suggesting new approaches to cancer control.

Genetic and epigenetic changes in stomach cancer.

Genetic and epigenetic alterations of multiple cancer-related genes and molecules are implicated in the development and progression of human gastric carcinomas. Reactivation of telomerase, inactivation of p53 tumor suppressor gene, overexpression of cyclin E, and reduced expression of p27 KIP1 by disorganized degradation in proteasome are common events of both well-differentiated and poorly differentiated gastric adenocarcinomas. Inactivation of hMLH1 mismatch repair gene by CpG hypermethylation resulting in microsatellite instability, amplification of c-erbB2 oncogene, inactivation of APC tumor suppressor gene, and K-ras mutations are preferentially associated with well-differentiated gastric cancer. Conversely, reduction or loss of E-cadherin and catenins by both mutation and CpG hypermethylation and K-sam and c-met oncogene amplification are necessary for the development and progression of poorly differentiated or scirrhous gastric carcinomas. Interaction between cancer cells expressing c-met and hepatocyte growth factor from stromal cells is implicated in morphogenesis of gastric cancer.

Telomerase activity in preneoplastic and neoplastic gastric and colorectal lesions.

Recent evidence indicates that telomerase activity may be necessary for cell immortality, which is required for the sustained and indefinite growth of most malignant cells. We analyzed telomerase activity in gastric and colorectal cancers and in gastric and colorectal precancerous lesions to determine whether malignant progression depends on the activation of telomerase and at what stage of carcinogenesis cells have detectable telomerase activity. Telomerase activity was measured by the telomeric repeat amplification protocol assay and was detected in 17 (85%) of 20 primary gastric carcinoma tissues and in 19 (95%) of 20 primary colorectal carcinomas, regardless of tumor staging and histological types. All nodal metastases, peritoneal metastases, and a recurrent gastric cancer tumor were positive. All cell lines established from gastric and colorectal cancers contained telomerase activity. In precancerous lesions, 10 (100%) of 10 colorectal tubular adenomas were telomerase positive, in addition to 3 (23%) of 13 gastric intestinal metaplasias and 1 (50%) of 2 gastric adenomas, whereas the corresponding gastric normal mucosas as well as colorectal mucosas were negative. These results indicate overall that reactivation of telomerase may occur at an early stage of carcinogenesis and may correlate well with malignant progression of gastric cancer. Telomerase activity thus may serve as a powerful additional tool for cancer diagnosis.

Site-selective 8-Cl-cAMP which causes growth inhibition and differentiation increases DNA (CRE)-binding activity in cancer cells.

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site-selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP-dependent protein kinase type II regulatory subunit, RII beta. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA-binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS-174T), gastric cancer (TMK-1), and leukemia (K-562)] and rodent pheochromocytoma (PC12) show a concentration-dependent increase in binding activity to a synthetic DNA that contained the cAMP-responsive element 5'-TGACGTCA-3' after treatment with 8-Cl-cAMP. Such an increase in cAMP-responsive element binding activity was not observed in the 8-C1-cAMP-unresponsive MKN-1 gastric cancer cells. These findings indicate that the antitumor activity of site-selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.

Identification of concurrent germ-line mutations in hMSH2 and/or hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds.

We analyzed microsatellite instability, alterations of the polyadenine tract in TGF-beta RII (transforming growth factor beta type II receptor gene), and mutations of hMSH2 and hMLH1 in 32 patients with familial colorectal cancer (29 kindreds) fulfilling the clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC), defined at the 34th Annual Meeting of Japanese Society for Cancer of the Colon and Rectum (Tokushima, Japan, 1991), including five kindreds fulfilling the Amsterdam criteria. Eighteen of 32 (56%) cases were replication error positive (RER+) at two or more microsatellite loci analyzed. The clinicopathological characteristics of RER+ cases corresponded well with those reported previously. Eleven of 18 RER+ cases showed RER+ at most of the microsatellite loci examined. Among these 11 cases (10 kindreds), 3 kindreds fulfilled the Amsterdam criteria and 7 kindreds did not. For these 10 kindreds, germ-line mutations in hMSH2 and hMLH1 were detected for 6 kindreds by PCR-SSCP analysis and direct sequencing. Only two of these six fulfilled the Amsterdam criteria; more than one germ-line mutation was detected in hMSH2 and/or hMLH1. Specifically, two point mutations of hMSH2 were detected in two kindreds, one point mutation of both hMSH2 and hMLH1 was detected in one kindred, two point mutations of hMSH2 and one point mutation of hMLH1 were detected in one kindred, and two point mutations of hMLH1 and one point mutation of hMSH2 were detected in one kindred. In addition, 19 of 26 (74%) cancer lesions of these 11 cases with the RER phenotype showed alterations of the polyadenine tract in TGF-beta RII. From our data, although seven kindreds did not fulfill the Amsterdam criteria, we considered them as HNPCC. Therefore, we suggest that the "Japanese criteria" have the advantage of being able to detect more HNPCC kindreds from borderline HNPCC kindreds.

Telomerase activity in premalignant and malignant lesions of human oral mucosa.

Recent studies have demonstrated a strong association between carcinogenesis and re-activation of telomerase in various human tumors. In the present study, we have analyzed the telomerase activity in 105 oral mucosal samples, including normal mucosa and premalignant and malignant lesions, by using the telomeric repeat amplification protocol assay. The telomerase activity was detected in normal oral squamous epithelium and in 75% of the oral leukoplakias and oral carcinomas. Although the telomerase activity was observed in normal and hyperplastic squamous epithelium, it showed some relationship with certain clinico-pathological factors in malignant lesions. Telomerase activity was found to have a relationship with the grade of tumor differentiation. Of 34 well-differentiated squamous cell carcinomas, only 10 (30%) exhibited high telomerase activity, whereas in moderately or poorly differentiated squamous cell carcinomas, all seven (100%) tumors displayed high activity. In addition, the level of telomerase activity had an inverse correlation with the treatment response in the early-stage tumors, and the activity differed significantly between the tumors in the following intraoral sites: nonkeratinizing mucosa (buccal mucosa, alveolus, and floor of mouth) and tongue. This preliminary result shows that telomerase activity is present in normal oral squamous epithelium, as it is in normal hematopoietic cells and in carcinomas, and that telomerase activity has a relationship with degree of tumor differentiation and treatment response. Thus, assessing the telomerase activity may be a useful prognostic marker in oral squamous cell carcinomas.

High frequency of p53 gene mutation in adenocarcinomas of the gallbladder.

p53 mutations in adenocarcinomas of the gallbladder were analyzed by deoxynucleotide sequencing of the gene. Of 23 cases, 16 (70%) harbored 18 missense mutations in exon 5, 6, or 8 of the p53 gene. The characteristics of the p53 mutation spectrum observed in gallbladder carcinomas were (a) frequent mutations at an A:T pair [10 (55%) of 18 mutations], (b) high transversion incidence [12 (66%) of 18 mutations], and (c) only one mutation at the CpG site. The immunohistochemical study revealed that 36 (55%) of 65 cases showed an abnormal accumulation of p53 immunoreactivity, and 12 (52%) of 23 cases had p21 expression. No statistical correlation was observed between p53 and p21 immunoreactivity. These results suggest that p53 mutations may confer the carcinogenesis of the adenocarcinoma of the gallbladder with the specific mutation spectrum of p53.

Effect of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (vesnarinone) on the growth of gastric cancer cell lines.

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone ) is a synthetic oral cardiotonic agent that has been used for the treatment of patients with congestive heart failure. Six days of treatment with 30 microg/ml of vesnarinone induced 20-80% growth inhibitions in five out of six gastric carcinoma cell lines examined. Cell cycle analysis revealed that the vesnarinone-sensitive TMK-1 gastric cancer cell line exhibited a significant G0-G1 arrest without evidence of apoptotic cell death induction after 48 h of treatment. Interestingly, this phenomenon was preceded by a marked reduction in the expression of cyclin A, D1 and E as well as cyclin-dependent kinase 2 (CDK2). On the other hand, no significant change was observed in the expression of p21(Waf1/Cip1), p27Kip1 nor various growth factors and their receptor genes. Overall these results indicate that vesnarinone inhibits the growth of gastric cancer cells by down-regulating G1 cyclins and CDK2 to induce G0-G1 arrest through a pathway different from that of cyclin inactivation by p21(Waf1/Cip1) or p27Kip1.

Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines.

Retinoic acid (RA) has been shown to be effective in suppressing premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck. However, the precise mechanisms of these effects are still uncertain. In the present study, we examined the effect of 9-cis-RA on the growth of six oral cancer cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-N-1 and Ho-1-u-1). In addition, the relationship among growth and differentiation of tumor cells, RA responsiveness and the expression of nuclear retinoic acid receptors were also investigated. Among the six cell lines examined, five (HSC-2, HSC-3, HSC-4, Ca9-22 and Ho-1-u-1) displayed growth inhibition after treatment with 1x10(-6) M 9-cis-RA, while Ho-1-N-1 cells were resistant to 9-cis-RA. The expression level of RARbeta in 9-cis-RA resistant Ho-1-N-1 cells was very low in comparison with the sensitive cell lines. On the other hand, all of the six the cell lines expressed RARalpha, RARgamma, and RXRalpha at various levels. 9-cis-RA induced accumulation of cell population in G1 phase in HSC-3 cells on the 6th day of the treatment, followed by a marked reduction in the levels of hyperphosphorylated pRB, whereas p53 level was not altered. Interestingly, 9-cis-RA induced transiently the expression of p21(Waf1/Cip1), p27(Kip1), p300, CBP, BAX, Bak and bcl-2 proteins, respectively. This effect was associated with reduction of cyclin D1, cdk4 and CDK-activating kinase (cyclin H and cdk7) protein in HSC-3 cells. These results suggest that the growth inhibitory effect of 9-cis-RA on oral squamous cell carcinoma may depend on the expression levels of RARs, especially RARbeta proteins and RXRalpha proteins, and that 9-cis-RA may provide a powerful therapeutic agent for head and neck cancers.