Gene expression profile of ABC transporters and cytotoxic effect of ibuprofen and acetaminophen in an epithelial ovarian cancer cell line in vitro

PURPOSES: To determine the basic expression of ABC transporters in an epithelial ovarian cancer cell line, and to investigate whether low concentrations of acetaminophen and ibuprofen inhibited the growth of this cell line in vitro. METHODS: TOV-21 G cells were exposed to different concentrations of acetaminophen (1.5 to 15 μg/mL) and ibuprofen (2.0 to 20 μg/mL) for 24 to 48 hours. The cellular growth was assessed using a cell viability assay. Cellular morphology was determined by fluorescence microscopy. The gene expression profile of ABC transporters was determined by assessing a panel including 42 genes of the ABC transporter superfamily. RESULTS: We observed a significant decrease in TOV-21 G cell growth after exposure to 15 μg/mL of acetaminophen for 24 (p=0.02) and 48 hours (p=0.01), or to 20 μg/mL of ibuprofen for 48 hours (p=0.04). Assessing the morphology of TOV-21 G cells did not reveal evidence of extensive apoptosis. TOV-21 G cells had a reduced expression of the genes ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 within the ABC transporter superfamily. CONCLUSIONS: This study provides in vitro evidence of inhibitory effects of growth in therapeutic concentrations of acetaminophen and ibuprofen on TOV-21 G cells. Additionally, TOV-21 G cells presented a reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 transporters.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8% (TRAIL) or 17% (ADM) to 38.7%, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG-100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitroand the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

NFAT transcription factors: from cell cycle to tumor development

The nuclear factor of activated T cells (NFAT) family of transcription factors has been primarily identified in immune cells; however, these proteins have been recently found to be functionally active in several other non-immune cell types. NFAT proteins are activated upon different stimuli that lead to increased intracellular calcium levels. Regardless of their widely known cytokine gene expression properties, NFATs have been shown to regulate other genes related to cell cycle progression, cell differentiation and apoptosis, revealing a broader role for these proteins in normal cell physiology. Several reports have addressed the participation of NFATs in many aspects of malignant cell transformation and tumorigenic processes. In this review, we will discuss the involvement of the different NFAT family members in the regulation of cell cycling, differentiation and tumor formation, and also its implications on oncogenesis. Better understanding the mechanisms by which NFATs regulate cell cycle and tumor-related events should be relevant for the development of rational anti-cancer therapies.

Profile of thyroid hormones in breast cancer patients

Estrogen involvement in breast cancer has been established; however, the association between breast cancer and thyroid diseases is controversial. Estrogen-like effects of thyroid hormone on breast cancer cell growth in culture have been reported. The objective of the present study was to determine the profile of thyroid hormones in breast cancer patients. Serum aliquots from 26 patients with breast cancer ranging in age from 30 to 85 years and age-matched normal controls (N = 22) were analyzed for free triiodothyronine (T3F), free thyroxine (T4F), thyroid-stimulating hormone (TSH), antiperoxidase antibody (TPO), and estradiol (E2). Estrogen receptor ß (ERß) was determined in tumor tissues by immunohistochemistry. Thyroid disease incidence was higher in patients than in controls (58 vs 18%, P < 0.05). Subclinical hyperthyroidism was the most frequent disorder in patients (31%); hypothyroidism (8%) and positive anti-TPO antibodies (19%) were also found. Subclinical hypothyroidism was the only dysfunction (18%) found in controls. Hyperthyroidism was associated with postmenopausal patients, as shown by significantly higher mean T3 and T4 values and lower TSH levels in this group of breast cancer patients than in controls. The majority of positive ERß tumors were clustered in the postmenopausal patients and all cases presenting subclinical hyperthyroidism in this subgroup concomitantly exhibited Erß-positive tumors. Subclinical hyperthyroidism was present in only one of 6 premenopausal patients. We show here that postmenopausal breast cancer patients have a significantly increased thyroid hormone/E2 ratio (P < 0.05), suggesting a possible tumor growth-promoting effect caused by this misbalance.

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 µg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 µg/mL, respectively. A high concentration of ethyl acetate extract (100 µg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 µg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42%), 1,2,4-tripropylbenzene (13.09%), 9-cedranone (12.75%), ledene oxide (7.22%), caryophyllene (1.82%), and caryophyllene oxide (1.15%). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

In vitro and in vivo studies of pirarubicin-loaded SWNT for the treatment of bladder cancer

Intravesical chemotherapy is an important part of the treatment for superficial bladder cancer. However, the response to it is limited and its side effects are extensive. Functional single-walled carbon nanotubes (SWNT) have shown promise for tumor-targeted accumulation and low toxicity. In the present study, we performed in vivo and in vitro investigations to determine whether SWNT-based drug delivery could induce high tumor depression in rat bladder cancer and could decrease the side effects of pirarubicin (tetrahydropyranyl-adriamycin, THP). We modified SWNT with phospholipid-branched polyethylene glycol and constructed an SWNT-THP conjugate via a cleavable ester bond. The cytotoxicity of SWNT-THP against the human bladder cancer cell line BIU-87 was evaluated in vitro. Rat bladder cancer in situ models constructed by N-methyl-N-nitrosourea intravesical installation (1 g/L, 2 mg/rat once every 2 weeks for 8 weeks) were used for in vivo evaluation of the cytotoxicity of SWNT and SWNT-THP. Specific side effects in the THP group including urinary frequency (N = 12), macroscopic hematuria (N = 1), and vomiting (N = 7) were identified; however, no side effects were observed with SWNT-THP treatment. Flow cytometry was used to assess the cytotoxicity in vitro and in vivo. Results showed that SWNT alone did not yield significant tumor depression compared to saline (1.74 ± 0.56 and 1.23 ± 0.42%) in vitro. SWNT-THP exhibited higher tumor depression than THP-saline in vitro (74.35 ± 2.56 and 51.24 ± 1.45%) and in vivo (52.46 ± 2.41 and 96.85 ± 0.85%). The present findings indicate that SWNT delivery of THP for the treatment of bladder cancer leads to minimal side effects without loss of therapeutic efficacy. Therefore, this nanotechnology may play a crucial role in the improvement of intravesical treatment of bladder cancer.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Clinicopathological significance of PTPN12 expression in human breast cancer

Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a recently identified tumor suppressor gene (TSG) that is frequently compromised in human triple-negative breast cancer. In the present study, we investigated the expression of PTPN12 protein by patients with breast cancer in a Chinese population and the relationship between PTPN12 expression levels and patient clinicopathological features and prognosis. Additionally, we explored the underlying down-regulation mechanism from the perspective of an epigenetic alteration. We examined PTPN12 mRNA expression in five breast cancer cell lines using semi-quantitative reverse-transcription PCR, and detected PTPN12 protein expression using immunohistochemistry in 150 primary invasive breast cancer cases and paired adjacent non-tumor tissues. Methylation-specific PCR was performed to analyze the promoter CpG island methylation status of PTPN12. PTPN12 was significantly down-regulated in breast cancer cases (48/150) compared to adjacent noncancerous tissues (17/150; P < 0.05). Furthermore, low expression of PTPN12 showed a significant positive correlation with tumor size (P = 0.047), lymph node metastasis (P = 0.001), distant metastasis (P = 0.009), histological grade (P = 0.012), and survival time (P = 0.019). Additionally, promoter CpG island hypermethylation occurs more frequently in breast cancer cases and breast cancer cell lines with low PTPN12 expression. Our findings suggest that PTPN12 is potentially a methylation-silenced TSG for breast cancer that may play an important role in breast carcinogenesis and could potentially serve as an independent prognostic factor for invasive breast cancer patients.

A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.