Cell Testicular Cancer Research Articles

Raman Spectroscopy Can Detect and Monitor Cancer at Cellular Level: Analysis of Resistant and Sensitive Subtypes of Testicular Cancer Cell Lines

: Raman spectroscopy has been applied to analyze testicular cancer cell lines. Spectral differences between resistant and sensitive subtypes of testicular cancer cell line 833k samples were successfully analysed. The technique allowed reproducible and quantitative analysis of the specimen and illustrated the chemical specifications of the samples precisely. Six pairs of testicular cancer cell line 833k were studied and the findings were backed by statistical methods; that is, partial least squares discriminant analysis (PLS-DA). It was concluded that Raman spectroscopy can objectively differentiate between resistant and sensitive cell lines. These results suggest that in the future it may be possible to use cell lines and diagnostic Raman spectroscopy for preoperative classification of biological molecules. Further research is underway to determine whether results obtained from spectroscopic analysis of cell lines can be applied to actual human tissue samples.

Effects of curcumin on bleomycin-induced apoptosis in human malignant testicular germ cells

Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has attracted interest because of its anti-inflammatory and chemopreventive activities. However, no study has been carried out so far to elucidate its interaction with bleomycin in testicular cancer cells. In this study, we investigated the effects of curcumin and bleomycin on apoptosis signalling pathways and compared the effects of bleomycin with H2O2 which directly produces reactive oxygen species. We measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and Cyt-c levels in NCCIT cells incubated with curcumin (5 μM), bleomycin (120 μg/ml), bleomycin + curcumin, H2O2 (35 μM), and H2O2 + curcumin for 72 h. Curcumin, bleomycin, and H2O2 caused apoptosis indicated as increases in caspase-3, caspase-8, and caspase-9 activities and Bax and cytoplasmic Cyt-c levels and a decrease in Bcl-2 level. Concurrent use of curcumin with bleomycin decreased caspase activities and Bax and Cyt-c levels compared to their separate effects in NCCIT cells. Our findings suggest that concurrent use of curcumin during chemotherapy in testis cancer should be avoided due to the inhibitory effect of curcumin on bleomycin-induced apoptosis.

Involvement of the prostaglandin D2 signal pathway in retinoid-inducible gene 1 (RIG1)-mediated suppression of cell invasion in testis cancer cells

Retinoid-inducible gene 1 (RIG1), also called tazarotene-induced gene 3, belongs to the HREV107 gene family, which contains five members in humans. RIG1 is expressed in high levels in well-differentiated tissues, but its expression is decreased in cancer tissues and cancer cell lines. We found RIG1 to be highly expressed in testicular cells. When RIG1 was expressed in NT2/D1 testicular cancer cells, neither cell death nor cell viability was affected. However, RIG1 significantly inhibited cell migration and invasion in NT2/D1 cells. We found that prostaglandin D2 synthase (PTGDS) interacted with RIG1 using yeast two-hybrid screens. Further, we found PTGDS to be co-localized with RIG1 in NT2/D1 testis cells. In RIG1-expressing cells, elevated levels of prostaglandin D2 (PGD2), cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This indicated that RIG1 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated RIG1-mediated suppression of migration and invasion. These results suggest that RIG1 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Our study suggests that RIG1-PGD2 signaling might play an important role in cancer cell suppression in the testis.

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15-44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-L-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H(2)O(2) which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H(2)O(2) significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H(2)O(2) significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H(2)O(2). NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H(2)O(2).

Effects of curcumin on bleomycin-induced oxidative stress in malignant testicular germ cell tumors

Bleomycin is commonly used in the treatment of testicular cancer. Bleomycin generates oxygen radicals, induces the oxidative cleavage of DNA strands and induces cancer cell apoptosis. Curcumin (diferuloylmethane) is a potent antioxidant and chief component of the spice turmeric. No study investigating the effects of curcumin on intrinsic and bleomycin-induced oxidative stress in testicular germ cell tumors has been reported in the literature. For this reason, the present study aimed to examine the effects of curcumin on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testicular cancer cells incubated with bleomycin and the results were compared with cells treated with H2O2 which directly produces oxidative stress. The protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide (LPO) levels and total antioxidant capacity in the two testicular cancer cell lines were determined. Results showed that bleomycin and H2O2 significantly increased protein carbonyl, TBARS, 8-isoprostane and LPO levels in the NTera-2 and NCCIT cell lines. Bleomycin and H2O2 significantly decreased the antioxidant capacity and GSH levels in NTera-2 cells. Curcumin significantly decreased LPO, 8-isoprostane and protein carbonyl content, and TBARS levels increased in cells treated with bleomycin and H2O2. Curcumin enhanced GSH levels and the antioxidant capacity of NTera-2 cells. In conclusion, curcumin inhibits bleomycin and H2O2-induced oxidative stress in human testicular cancer cells.

Relationship of testicular cancer incidence and cellular phone use.

e12008 Background: Because of the ubiquitous usage of cellphones, any health consequences, if found, would be of paramount importance. Many have questioned the relationship of cellphone radiofrequency waves and malignancies such as brain tumors. Because men usually store their phones in close proximately to the testicles, we analyzed the relationship between cell phone usage and testicular cancer. Methods: Testicular cancer data was obtained from SEER-9 United States Population Data Base of the National Cancer Institute. Cell phone subscription data was obtained from the World Health Organization publication. Initial data analysis was done from 1991 to 2008, with subsequent age specific and other analyses performed from 1999 to 2008. Trends over time were calculated as Annual Percentage Change of incidence rates (APC) by using log linear models. Results: Rates of United States mobile subscriptions increased exponentially since the late 1990’s reaching 88.87 percent of the population by 2008. There was no statistically significant change in incidence rates of all testicular tumors (APC 0.7; CI -0.2, 0.7), Seminoma (APC 0.2; CI 0.8, 1.1) and non-Seminoma (APC 1.4; CI -0.6, 3.4) from 1999 to 2008. In the age specific analysis, three groups showed an increase in incidence rates: all testicular cancer age 25-34 (APC 2; CI 0.6, 3.4), All testicular cancer age greater than 45 (APC 1.7; CI 0.1, 3.3) and Seminoma age 25-34 (APC 2.1; CI 0.6, 3.7). All these changes were only minor percentage increase when compared to mobile subscriptions. Conclusions: Based on incidence data, there is no convincing evidence of increased risk of testicular cancer from cell phone use. Given possible lag time incidence, continued monitoring is needed.

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H(2)O(2) which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H(2)O(2) in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H(2)O(2) on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H(2)O(2), and/or NAC. We found half of the lethal dose (LD(50)) of bleomycin on NCCIT cell viability as 120 μg/ml after incubation for 72 h. Incubation with bleomycin (LD(50)) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10 mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.

Abstract 4910: Apoptotic effects of curcumin and bleomycin alone or in combination on malignant testicular germ cells

Abstract Aims: Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer, but side effects are common. Bleomycin increases oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in several types of malignancies. However, no study has been carried out so far to elucidate its possible anticancer activity and interaction with Bleomycin in testicular cancer cells. Methods: In this study, we investigated the effects of Curcumin and Bleomycin alone or in combination on apoptosis-signalling-pathways. We determined the cytotoxic effect of Bleomycin and Curcumin on NTera-2 (NT2) cells using 3-(4,5) dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and measured apoptosis signalling pathway markers such as Caspase-3, Caspase-8, Caspase-9 activities and Bcl-2, Bax and Cyt-c levels in NT2 cells incubated with Bleomycin in the presence or absence of Curcumin. Results: Median lethal dose (LD50) of Bleomycin on NT2 cell viability as 400 μg/ml after an incubation of 24 h and LD50 of Curcumin as 20 μM. Treatment with Bleomycin (400 μg/ml) and Curcumin (20 μM) alone for 24 h caused 3.2-fold and 3-fold increase in caspase-3 activity, 3.4-fold and 2-fold increase in caspase-8 activity, 2.9-fold and 2.8 fold increase in caspase-9 activities respectively. These treatments alone also caused 2-fold increase in Bax and 2-fold increase in cytoplasmic Cyt-c levels while leading 2-fold decrease in Bcl-2 levels. Combination of Curcumin with Bleomycin induced Caspase-3,-8,-9 activities more than their separate effects in NT2 cells. Specifically combination of Curcumin (20 μM) with Bleomycin (400 μg/ml) induced 4.2-fold increase in Caspase-3, 4.3-fold increase in caspase-8 activity, 4-fold increase in caspase-9 activity, 3-fold increase in cytoplasmic Cyt-c levels and 3-fold decrease in Bcl-2 levels.Conclusion: Bleomycin has proven to be an essential component of the cisplatin-based chemotherapy regimens, used in the treatment of testicular cancer. However, the use of Bleomycin is limited because of Bleomycin-induced pneumonitis, a complication occurring in approximately 10% of patients treated with Bleomycin and fatal in approximately 10% of the cases. Our observations suggest that the effects of Bleomycin on apoptosis-signalling-pathways were improved by Curcumin. Co-administration of Bleomycin with Curcumin could be a new strategy to use less dosage of Bleomycin and prevent patients from Bleomycin-induced side effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4910. doi:1538-7445.AM2012-4910

Abstract 1668: Understanding cancer associated SNPs in the TERT-CLPTM1L locus through population genetics

Abstract Telomeres form the ends of eukaryotic chromosomes and are vital in maintaining genetic integrity. TERT encodes the telomerase reverse transcriptase, which is responsible for maintaining telomere ends by addition of (TTAGGG)n nucleotide repeats at the telomere. Recent genome-wide association studies have found common genetic variants at theTERT-CLPTM1L locus (5p15.33) associated with increased risk of several cancers, including glioma, basal cell carcinoma, testicular, pancreatic, breast and lung cancers, and possibly overall cancer risk. Our prior studies of common single nucleotide polymorphisms (SNPs) in the TERT gene region found low differentiation among populations and higher than expected levels of haplotype diversity in data from the Human Genome Diversity Panel (HGDP) and the International HapMap Project. In the current study we significantly expanded the population genetic analyses by utilizing data from the 1000 Genomes Project (2011 October release). Genetic variation from a total of 1092 individuals from 14 populations with European, East Asian, West African or American ancestry was evaluated. Data were acquired for 3468 variants in a 206kb region on 5p15.33, which included the TERT-CLPTM1L gene and flanking regions. The GLU genetics’ ld.tagzilla module was used for the tag analysis with a linkage disequilibrium (LD) pairwise r2 threshold of 0.8. Arlequin (3.5) and Genepop (4.1) packages were used to estimate diversity, LD, heterozygosity, and differentiation (FST) among populations. Pairwise LD was analyzed separately for the 4 ancestral groups (European, East Asian, West African and American) and used to select tag SNPs for each region. The 4 ancestral groups had differing LD patterns with the lowest level of LD occurring in individuals from Africa, as expected. These data were used to create the appropriate haplotype-tagging SNPs for the TERT-CLPTM1L region in each population; we present recommended tagging SNPs by ancestral group. The TERT-CLPTM1L gene region had low nucleotide diversity (mean 2 x 10−4) and differentiation among ancestral groups (90% of loci in this region had FST <0.10). The limited diversity in this region may be due to its critical role in telomere maintenance and chromosomal stability. The recently identified cancer associated SNPs in this region had differing genotype frequencies among ancestral groups. Variation in cancer associated alleles among ancestral groups may correlate to varying disease risks. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1668. doi:1538-7445.AM2012-1668

Synergistic anticancer activity of curcumin and bleomycin: An in�vitro study using human malignant testicular germ cells

Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols used for testicular cancer; however, side-effects are common. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in a number of malignancies. However, to date no study has been carried out to elucidate its anticancer activity and interaction with bleomycin in testicular cancer cells. In this study, we investigated and compared the effects of curcumin, bleomycin and hydrogen peroxide (H2O2) on apoptotic signaling pathways. Curcumin (20 µM), bleomycin (400 µg/ml) and H2O2 (400 µM) incubation for 24 h decreased the viability of NTera-2 cells, and increased caspase-3, -8 and -9 activities, Bax and cytoplasmic cytochrome c levels and decreased Bcl-2 levels. The concurrent use of curcumin with bleomycin induced caspase-3, -8 and -9 activities to a greater extent in NTera-2 cells than the use of each drug alone. Our observations suggest that the effects of curcumin and bleomycin on apoptotic signaling pathways are synergistic. Therefore, we propose to use curcumin together with bleomycin to decrease its therapeutic dose and, therefore, its side-effects.

Phenylarsonic acid compounds with broad-spectrum and potent cytotoxic activity against human cancer cells.

The in vitro cytotoxic activity profile of nine novel phenylarsonic acid (CAS 98-05-5, PAA) compounds against 17 human cancer cell lines including (a) ovarian cancer cell lines ES-2, PA-1, CAOV-3, OVCAR-3, (b) testicular cancer cell lines Ntera-2, Tera-2, N2NICP, 833K, and 64CP, (c) multiple myeloma cell lines ARH77, HS-Sultan, RPMI-8226, and U266, and (d) acute lymphoblastic leukemia (ALL) cell lines NALM-6, MOLT-3, ALL-1, and RS4; 11, was determined by the MTT assay. The lead compounds, 2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine (PHI-370) and 2-methylthio-4-(4'-phenylarsonic acid)-aminopyrimidine (PHI-380) caused apoptotic death in all 17 cancer cell lines at low micromolar concentrations, as documented by TUNEL assays and confocal laser scanning microscopy. PHI-380 was also tested and found to be very active against primary tumor cells isolated from surgical biopsy specimens of 14 patients with therapy-refractory non-small cell lung cancer, breast cancer, colon cancer, lymphoma, hepatoblastoma, or Wilm's tumor as well. Because of their broad-spectrum and potent anticancer activity and ability to induce apoptosis in primary tumor cells from therapy-refractory cancer patients, PAA compounds such as PHI-370 and PHI-380 may provide the basis for effective salvage regimens for patients with recurrent cancer.

Lipophilic Pt(II) complexes with selective efficacy against cisplatin-resistant testicular cancer cells

A series of dichloridoplatinum(II) complexes with selective and high cytotoxicity [IC 90(96 h) ≤ 3 μM] against cisplatin-resistant 1411HP testicular cancer cells were identified. They bear stationary 6-aminomethylnicotinate or 2,4-diaminobutyrate ligands esterified with lipophilic terpenyl residues, i.e., (−)/(+)-menthyl, (+)-cedrenyl, (−)-menthoxypropyl, or with a decyl-tethered 1,1,2-triphenylethene. They accumulated to a larger extent in 1411HP cells than in cells of the cisplatin-sensitive H12.1 germ cell tumour. Their mechanism of apoptosis induction differed from that of cisplatin by being independent of p53 and of caspase-3 activation and by an early loss of the mitochondrial membrane potential. The new complexes are promising candidates for the treatment of cisplatin-resistant testicular tumours.

The Apoptotic Effect of Arsenic Compounds on MA-10 Mouse Leydig Tumor Cells.

Arsenic is an environmental toxicant. The research has shown that long term exposure can be harmful to human body, and has been associated with some disease, such as neuropathy, cardiovascular disease, diabetes, and hypertension. In addition to chronic diseases, in recent years, much evidence has revealed that long term arsenic exposure was associated with increased risks of some cancers, including skin, lung, bladder, and liver cancer, as well as others. However, some researchers surprisingly found that arsenic trioxide showed the good therapeutic effect on APL (acute promyelocytic leukemia) patients, and decrease APL cell viability. Because of the increasing incident rate of the testicular cancer, we assumed that whether arsenic might show the same efficiency in testicular cancer cells. To prove our assumption, we used MA-10 cells, a mouse Leydig tumor cell line, and treated with different concentration of sodium arsenite and dimethylarsenic acid. Our data showed that MA-10 cells appeared rounded-up and membrane blebbings in treatment with 100 µM sodium arsenite for 3 hours. Besides, MTT assay demonstrated that MA-10 cells also appeared cell death in treatment with 10 mM dimethylarsenic acid for 24 hours. In flow cytometry and western blot analysis, both treatments of sodium arsenite and dimethylarsenic acid can increase the ratio of subG1 phase and the level of the cleaved caspase-3 protein. Hence evidences showed that arsenic compounds can induce cell apoptosis not only in acute promyelocytic leukemia but also in MA-10 cells. These findings suggest that arsenic is the potential strategy of anti-cancer drug development in the testicular cancer. (poster)

Role of Human Endogenous Retroviral Long Terminal Repeats (LTRs) in Maintaining the Integrity of the Human Germ Line

Retroviruses integrate a reverse transcribed double stranded DNA copy of their viral genome into the chromosomal DNA of cells they infect. Occasionally, exogenous retroviruses infect germ cells and when this happens a profound shift in the virus host dynamic occurs. Retroviruses maintained as hereditable viral genetic material are referred to as endogenous retroviruses (ERVs). After millions of years of co-evolution with their hosts many human ERVs retain some degree of function and a few have even become symbionts. Thousands of copies of endogenous retrovirus long terminal repeats (LTRs) exist in the human genome. There are approximately 3000 to 4000 copies of the ERV-9 LTRs in the human genome and like other solo LTRs, ERV-9 LTRs can exhibit distinct promoter/enhancer activity in different cell lineages. It has been recently reported that a novel transcript of p63, a primordial member of the p53 family, is under the transcriptional control of an ERV-9 LTR [1]. The expression of different p63 transcript isoforms has been previously shown to have an important role in replenishing cutaneous epithelial stem cells and maintaining the fidelity of the female germ line [2]. In this recent report, a novel p63 transcript, designated GTAp63, is described as specifically expressed in healthy human testes and germ cell precursors of human testes but not in testicular cancer cells. The ability of ERV-9 regulatory regions to contribute to the maintenance of male germ line stability is yet another example of how ERVs have evolved to serve an important function in the physiology of their human hosts.

Serine/Threonine Kinase 17A Is a Novel p53 Target Gene and Modulator of Cisplatin Toxicity and Reactive Oxygen Species in Testicular Cancer Cells

Testicular cancer is highly curable with cisplatin-based therapy, and testicular cancer-derived human embryonal carcinoma (EC) cells undergo a p53-dominant transcriptional response to cisplatin. In this study, we have discovered that a poorly characterized member of the death-associated protein family of serine/threonine kinases, STK17A (also called DRAK1), is a novel p53 target gene. Cisplatin-mediated induction of STK17A in the EC cell line NT2/D1 was prevented with p53 siRNA. Furthermore, STK17A was induced with cisplatin in HCT116 and MCF10A cells but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element that binds endogenous p53 in a cisplatin-dependent manner was identified 5 kb upstream of the first coding exon of STK17A. STK17A is not present in the mouse genome, but the closely related gene STK17B is induced with cisplatin in mouse NIH3T3 cells, although this induction is p53-independent. Interestingly, in human cells containing both STK17A and STK17B, only STK17A is induced with cisplatin. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with the up-regulation of detoxifying and antioxidant genes, including metallothioneins MT1H, MT1M, and MT1X that have previously been implicated in cisplatin resistance. In addition, knockdown of STK17A resulted in decreased cellular reactive oxygen species, whereas STK17A overexpression increased reactive oxygen species. In summary, we have identified STK17A as a novel direct target of p53 and a modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells.

Abstract 177: Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin resistant human testicular carcinoma cells via the Fas/FasL pathway

Abstract A major role for p53 in the response and execution of apoptosis after chemotherapy has been described for many cancers. Although testicular cancer (TC) often express high levels of p53 and almost no TP53 mutations are found, resistance to chemotherapy is still observed in a subgroup of TC patients. Therefore, the involvement of wild-type p53 in cisplatin sensitivity and resistance of human TC is not clear. In the present study, we demonstrate that in resistant human TC cells (Tera2-CP, Scha, 2102EP) p53 resides in a complex with murine double minute 2 (MDM2) even at higher cisplatin concentrations compared to cisplatin sensitive human TC cells (Tera2). Hyper-activation of the p53 pathway using the MDM2 antagonist Nutlin-3 in TC cells leads to nuclear localization of p53 and effective apoptosis induction as single agent. Targeting MDM2 with short interfering RNA extremely sensitizes TC cells in a similar way. The observed effects are dependent on the presence of wild-type p53, since mutant p53 expressing TC cells (NCCIT) or wild-type p53 suppressed TC cells are resistant to Nutlin-3. Specifically, we show that the Fas death receptor pathway, i.e. enhanced Fas expression (3-fold) and the induction of Fas/FasL interactions, plays an important role in MDM2 antagonist-induced apoptosis in TC cells as demonstrated by using FasL blocking antibodies and FasL small interfering RNA. Importantly, we have identified a similar mechanism in Hodgkin lymphoma cells (KM-H2 and L540) and acute myeloid leukaemia cells (MOLM-13), suggesting a common profile for Nutlin-3-induced apoptosis, which involves the Fas death receptor pathway. Finally, we combined nutlin-3 with cisplatin, which strongly sensitized TC cells, including cisplatin-resistant TC cells, to apoptosis via Fas pathway activation and reduced cell survival more than 8-fold. These results indicate that targeting the p53/MDM2 axis, in combination with standard treatment, appears to be a powerful strategy to pursue in cisplatin-resistant or -refractory testicular cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 177. doi:10.1158/1538-7445.AM2011-177

Disruption of the MDM2–p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy

In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three microRNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes

TAp63, but not its homolog p53, eliminates oocytes that suffered DNA damage. An equivalent gene for guarding the male germ line is currently not known. Here we identify hitherto unknown human p63 transcripts with unique 5'-ends derived from incorporated exons upstream of the currently mapped TP63 gene. These unique p63 transcripts are highly and specifically expressed in testis. Their most upstream region corresponds to a LTR of the human endogenous retrovirus 9 (ERV9). The insertion of this LTR upstream of the TP63 locus occurred only recently in evolution and is unique to humans and great apes (Hominidae). A corresponding p63 protein is the sole p63 species in healthy human testis, and is strongly expressed in spermatogenic precursors but not in mature spermatozoa. In response to DNA damage, this human male germ-cell-encoded TAp63 protein (designated GTAp63) is activated by caspase cleavage near its carboxyterminal domain and induces apoptosis. Human testicular cancer tissues and cell lines largely lost p63 expression. However, pharmacological inhibition of histone deacetylases completely restores p63 expression in testicular cancer cells (>3,000-fold increase). Our data support a model whereby testis-specific GTAp63 protects the genomic integrity of the male germ line and acts as a tumor suppressor. In Hominidae, this guardian function was greatly enhanced by integration of an endogenous retrovirus upstream of the TP63 locus that occurred 15 million years ago. By providing increased germ-line stability, this event may have contributed to the evolution of hominids and enabled their long reproductive periods.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma

BackgroundStem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells.ResultsThe pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance.ConclusionsOur data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer.