Synergistic antitumor effect of ionomycin and cisplatin against renal cell carcinoma in vitro and in vivo

Objectives. To characterize the synergistic antitumor effects of the calcium ionophore, ionomycin, and of cisplatin against human renal cell carcinoma cell line, ACHN, both in vitro and in vivo.Methods. The in vitro growth rate of ACHN after exposure to these compounds was measured, using the MTT assay. The apoptotic features in ACHN were evaluated by DNA ladder analysis and flow cytometric analysis. Bcl-2 and Bax expression levels in ACHN after treatment were examined by Western blot. The synergistic antitumor effects of ionomycin and cisplatin against the growth of established ACHN tumors in athymic nude mice were then tested.Results. The in vitro growth rate of ACHN was suppressed more by ionomycin and cisplatin in combination than by either alone. DNA ladder and fragmentation were more obvious when the cells were incubated with ionomycin and cisplatin together than with either reagent alone. Ionomycin treatment increased the expression level of Bax protein, whereas Bcl-2 expression was not influenced. Although an intraperitoneal injection of cisplatin or an intratumoral injection of ionomycin against subcutaneous ACHN tumors somewhat reduced tumorigenicity in nude mice, the effect was significantly enhanced by a combination of these drugs.Conclusions. The synergistic antitumor effects suggest that ionomycin-based therapy could be a novel therapeutic strategy with which to treat advanced renal cell carcinoma.     

Verotoxin induces rapid elimination of human renal tumor xenografts in SCID mice

PURPOSE: Verotoxins (VTs) are subunit toxins produced by enteropathogenic Escherichia coli. The VT receptor glycolipid Gb3, which mediates the cytotoxicity of VTs, has been reported to be elevated on the surface of several tumor cell lines. In this study the effect of VT1 as an antineoplastic agent was assessed using various human urological cancer cell lines. MATERIALS AND METHODS: The expression of Gb3 on human cancer cell lines originating from renal cell carcinoma (ACHN, A-704, CAKI-1 and CAKI- 2), prostate cancer (LNCaP and PC3) and testicular tumor (2102Ep) were examined by FACScan (Becton Dickinson, Sunnyvale, California). These cell lines were cultured with various concentrations of VT1 and subjected to microculture tetrazolium dye assay for determination of cell viability. Furthermore, ACHN cells were inoculated into the backs of SCID mice and intratumor injection of VT1 was performed. Pathological samples were examined by hematoxylin and eosin staining as well as by TUNEL assay. RESULTS: The growth of ACHN, CAKI-1, A-704, 2102Ep and LNCaP but not CAKI-2 and PC3 was significantly inhibited by co-incubation with VT1, as determined by microculture tetrazolium dye assays, consistent with FACScan results for Gb3 expression. When mice bearing ACHN tumors were injected with VT1, rapid reduction in the size of subcutaneous tumors was observed with complete regression within 5 to 7 days. Pathological examination by the TUNEL method indicated that the cytotoxicity of VT1 was mediated by apoptosis. CONCLUSIONS: These results suggest that VTs could be candidates for antineoplastic agents against Gb3 expressing tumors for clinical use.     

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 10⁷ G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation. Presented in part at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Introducing the clusterin gene into human renal cell carcinoma cells enhances their metastatic potential

PURPOSE: We recently reported a protective role of clusterin expression against apoptosis induced by a wide variety of stimuli in several human cancer models. In the current study we tested the hypothesis that clusterin over expression confers a benefit for the metastasis of renal cell carcinoma through the inhibition of apoptosis induced by the various obstacles the cancer cells may confront after detachment from their primary origin. MATERIALS AND METHODS: We introduced clusterin complementary DNA into human renal cell carcinoma ACHN cells, which do not express detectable level of clusterin expression, and generated the clusterin over expressing cell line ACHN/CL and the control vector only transfected cell line ACHN/C. In vitro anti-cell death activity under anchorage independent conditions among ACHN sublines was examined by limiting dilution assay and cell survival assay in suspension. To investigate the in vivo effects of clusterin over expression on metastatic potentials each cell line was injected into the tail vein or renal subcapsule of nonobese diabetic, severe combined immunodeficient mice and the metastatic features in all abdominal and thoracic organs were evaluated. RESULTS: ACHN/CL showed significantly enhanced growth in limiting dilution cultures compared with ACHN/C. The analysis of cell survival in the floating assay also revealed that ACHN/CL had a powerful survival advantage in suspension compared with ACHN/C. Furthermore, ACHN/CL formed more than 5-fold as many metastatic nodules in the lung after intravenous injection than ACHN/C. Similarly more marked lung metastasis was observed after implanting ACHN/CL cells into the renal subcapsule than after implanting ACHN/C cells. In contrast, there were no significant differences among ACHN sublines in the growth rates in vitro and in vivo, cell motility or invasive ability. CONCLUSIONS: These findings suggest that, if clusterin is over expressed, it prolongs cell survival under unfavorable conditions in the metastatic process, resulting in the enhanced metastatic potential of renal cell carcinoma.     

Oncolytic viral gene therapy for prostate cancer using two attenuated,replication-competent,genetically engineered herpes simplex viruses

BACKGROUND: Attenuated, replication-competent herpes simplex virus mutants offer an exciting new modality in cancer therapy through their ability to selectively replicate within and kill malignant cells with minimal harm to normal tissues. METHODS: This study investigates the efficacy of two such viruses, G207 and NV1020, in human prostatic carcinoma. In vitro studies were performed on four human prostatic carcinoma cell lines, and in vivo single/multiple dose studies were undertaken on mice by using two human cell types. Tumor volume, histopathology at necropsy, and serum prostate specific antigen (PSA) were used as measures of antiproliferative effect in the in vivo experiments. RESULTS: Both viruses were effective in producing cytolytic effects in vitro at various multiplicities of infection in all cell lines tested. Both viruses demonstrated antitumor effects in vivo with a statistically significant decrease in serum PSA and inhibition of growth of both PC-3 and C4-2 subcutaneous xenografts. Tumor-free animals at necropsy were observed in the treated groups but not in control animals. CONCLUSION: These results display impressive activity against human prostate cancer and offer promise for the use of this modality in the future.     

Human renal cell cancer proliferation in tissue culture is tonically inhibited by opioid growth factor

PURPOSE: Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. MATERIALS AND METHODS: Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. RESULTS: OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. CONCLUSION: OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.     

苦参碱对人ACHN肾癌裸鼠皮下移植瘤的生长抑制作用

目的探讨苦参碱对人ACHN肾癌裸鼠皮下模型的生长抑制作用。方法皮下包埋法建立人ACHN肾癌裸鼠皮下移植瘤模型,按照随机原则将裸鼠分为50、75、100mg/kg苦参碱组和生理盐水组,各组均采用腹腔注射给药,5次/周,连续3周。每天测量肿瘤最长径和最短经,计算肿瘤体积,比较各组肿瘤生长抑制率;光镜及电镜观察苦参碱作用后肿瘤细胞形态改变。结果 35只裸鼠成瘤27只,观察满3周后,50、75、100mg/kg苦参碱组的肿瘤生长抑制率分别为19.51%、38.73%、46.08%,光镜下见苦参碱给药组不同程度的细胞坏死,且坏死范围随苦参碱浓度的增大而增大;电镜观察发现苦参碱作用后出现典型的细胞凋亡和坏死形态表现。结论苦参碱对人ACHN肾癌具有明显的生长抑制作用,这为临床应用苦参碱治疗肾癌提供了实验基础。     

Newcastle disease virus selectively kills human tumor cells.

Newcastle disease virus (NDV), strain 73-T, has previously been shown to be cytolytic to mouse tumor cells. In this study, we have evaluated the ability of NDV to replicate in and kill human tumor cells in culture and in athymic mice. Plaque assays were used to determine the cytolytic activity of NDV on six human tumor cell lines, fibrosarcoma (HT1080), osteosarcoma (KHOS), cervical carcinoma (KB8-5-11), bladder carcinoma (HCV29T), neuroblastoma (IMR32), and Wilm's tumor (G104), and on nine different normal human fibroblast lines. NDV formed plaques on all tumor cells tested as well as on chick embryo cells (CEC), the native host for NDV. Plaques did not form on any of the normal fibroblast lines. To detect NDV replication, virus yield assays were performed which measured virus particles in infected cell culture supernatants. Virus yield increased 10,000-fold within 24 hr in tumor and CEC supernatants. Titers remained near zero in normal fibroblast supernatants. In vivo tumoricidal activity was evaluated in athymic nude Balb-c mice by subcutaneous injection of 9 x 10(6) tumor cells followed by intralesional injection of either live or heat-killed NDV (1.0 x 10(6) plaque forming units [PFU]), or medium. After live NDV treatment, tumor regression occurred in 10 out of 11 mice bearing KB8-5-11 tumors, 8 out of 8 with HT-1080 tumors, and 6 out of 7 with IMR-32 tumors. After treatment with heat-killed NDV no regression occurred (P less than 0.01, Fisher's exact test). Nontumor-bearing mice injected with 1.0 x 10(8) PFU of NDV remained healthy. These results indicate that NDV efficiently and selectively replicates in and kills tumor cells, but not normal cells, and that intralesional NDV causes complete tumor regression in athymic mice with a high therapeutic index.     

Differences in cell kinetic changes among renal cancer cell lines treated with interferon-α

BACKGROUND: Interferon (IFN)-alpha shows certain clinical effects on the treatment of renal cell carcinoma. The purpose of the present study was to investigate its direct effects and to compare the responses among different human renal cancer cell lines. METHODS: Three cell lines, ACHN, RCC10RGB and OS-RC-2, were incubated with IFN-alpha and evaluated using MTT assay for cell proliferation and two-color flow cytometry for cell-cycle-specific cyclin expressions coupled with DNA ploidy analysis. RESULTS: Interferon-alpha inhibited cell proliferation and caused cell accumulation at S and G2/M phases. However, IFN-alpha induced no significant change in cyclins D1, E, A or B1 expression. Interestingly, cell kinetic changes caused by IFN-alpha were different among cell lines. Cell proliferation was suppressed most in ACHN, then RCC10RGB and least in OS-RC-2. Comparing DNA histograms, ACHN showed distinct increase of G2/M cells associated with elevation of late S cells. RCC10RGB showed a predominant increase of whole S cells accompanied with a slight increase of G2/M. OS-RC-2 showed a modest increase of S cells with a little change of G2/M cells. Chronological observation revealed that S-phase increase and proliferative inhibition appeared on day 1 and day 3, respectively, in ACHN and RCC10RGB, and on day 5 in OS-RC-2. CONCLUSIONS: Interferon-alpha induced substantial cell kinetic interference directly in the tested human renal carcinoma cell lines. The degree of change was different according to the nature of the cell line. It may partly indicate the variety of the efficacy of IFN-alpha treatment against renal cancers.     

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

Cancer gene therapy using a replication-competent herpes simplex virus type 1 vector.

OBJECTIVE: The authors investigate the efficacy of hrR3, a viral vector derived from herpes simplex virus type 1 (HSV 1), in destroying colon carcinoma cells in vitro and in vivo. The effect of adding the prodrug ganciclovir in combination with hrR3 infection also is assessed. SUMMARY BACKGROUND DATA: Most cancer gene therapy strategies use viral vectors that are incapable of replication. The HSV 1 vector hrR3 is capable of replication, and its replication is cytotoxic to cells. hrR3 also possesses the HSV-thymidine kinase gene, which converts ganciclovir into a toxic metabolite. Thus, the addition of ganciclovir to hrR3-infected cells may enhance the ability of hrR3 to destroy tumor cells. To increase specificity for tumor cells, hrR3 has a mutated ribonucleotide reductase gene and replicates selectively in cells with high levels of endogenous rbonucleotide reductase. Actively dividing cells such as tumor cells have high levels of endogenous ribonucleotide reductase for synthesis of DNA precursors. The authors are interested in the use of HSV 1 vectors to treat liver metastases from colorectal cancer. METHODS: Ribonucleotide reductase expression in several colon carcinoma cell lines and in primary cultures of human hepatocytes was determined by Western blot analysis. hrR3-mediated cytotoxicity in the colon carcinoma cell lines was determined using an in vitro assay. The human colon carcinoma cell line HT29 was injected into the flanks of nude mice followed by intratumoral injection of hrR3. Tumor growth rate was assessed with and without the addition of intraperitoneal ganciclovir. RESULTS: Ribonucleotide reductase levels in colon carcinoma cell lines are much higher than in primary cultures of human hepatocytes. hrR3 efficiently destroys colon carcinoma cell lines in vitro. A single intratumoral injection of hrR3 into HT29 flank tumors significantly reduces tumor growth rate, and the administration of ganciclovir has no additive effect. CONCLUSIONS: The inherent cytotoxicity of hrR3 replication effectively destroys colon carcinoma cells in vitro and in vivo. This cytotoxicity is not enhanced in vivo by the addition of ganciclovir. In the future, more efficacious and selective HSV 1 vectors may be useful in the treatment of cancer.     

Effects of tamoxifen and somatostatin analogue on growth of human medullary, follicular, and papillary thyroid carcinoma cell lines: tissue culture and nude mouse xenograft studies

The knowledge that (1) the normal thyroid contains somatostatin, (2) polypeptide growth factors influence thyroid cell function, and (3) thyroid cells contain steroid hormone receptors prompted us to add somatostatin analogue No. 201-995 (SMS) (5 ng/ml) and/or tamoxifen citrate (TAM) (5 mumol/L) to 7-day monolayer cultures (50,000 cells/well) of three separate human thyroid carcinoma cell lines: DR081 (medullary), WR082 (follicular), and NPA'87 (papillary). Results, tabulated as cell numbers/well (X10(5) on day 7, revealed that TAM inhibited growth of medullary and follicular cells and that TAM plus SMS inhibited growth of papillary cells. In vivo studies of subcutaneous tumor cell xenografts in nude mice have documented that TAM (5 mg subcutaneous pellet) significantly inhibits the growth of medullary implants. Flow cytometric DNA studies of medullary cell cultures demonstrated a reduced G2 + M phase with TAM treatment. For papillary cell implants, TAM plus SMS (5 micrograms subcutaneously, twice daily) did not suppress tumor growth. All three cell lines were negative for estrogen receptor; addition of estradiol (5 ng/ml) to medullary cell cultures neither stimulated replication nor reversed the inhibitory effects of TAM in vitro. We conclude that (1) TAM slowed the growth of a cell line of human medullary carcinoma, both in vitro and in vivo; (2) this effect was not reversed by estradiol; (3) TAM plus SMS inhibited replication of a papillary carcinoma cell line in vitro, but not in vivo; and (4) TAM alone and TAM plus SMS inhibited replication of cultures of a human follicular thyroid carcinoma cell line. TAM and SMS may be useful in treatment of some human thyroid carcinomas.     

Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma

OBJECTIVE: To determine whether alpha-fetoprotein (AFP)-regulated ribonucleotide reductase (RR) production would promote more vigorous and specific viral killing in AFP-expressing hepatocellular carcinoma (HCC). BACKGROUND: AFP is expressed in over 70% of primary HCC but not in normal adult liver. AFP production by HCC can be exploited to target viral killing of tumor cells. G207 is an oncolytic herpes virus lacking UL39, the gene encoding RR. RR is an enzyme required for viral DNA synthesis and cytotoxicity. METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed for AFP levels on Hep3B and PLC5 human HCC cells. An AFP-albumin enhancer-promoter complex (AFP-alb) was constructed in a luciferase vector to assess function. AFP-alb was cloned upstream of UL39 (AFP-alb/UL39) and transfected into HCC cells for G207 cytotoxicity assays. Viral plaque forming assays evaluated G207 replication. Hep3B flank tumors, with and without AFP-alb/UL39 transfection, were established in athymic mice (n = 28) and treated with G207. RESULTS: Hep3B had 5-fold higher AFP levels than PLC5 (P < 0.00001). AFP-alb increased luciferase expression 72-fold in Hep3B (P < 0.001) and 3-fold in PLC5 (P < 0.001). AFP-alb/UL39 transfection increased G207 cytotoxicity 93% in Hep3B (P < 0.0005), with no significant increase in PLC5. Peak viral titers were 46-fold higher in Hep3B transfected with AFP-alb/UL39 versus mock-transfected cells (P < 0.01), with no significant change in PLC5. Flanks tumors transfected with AFP-alb/UL39 and treated with G207 demonstrated a 76% volume reduction versus mock-transfected tumors infected with G207 (P < 0.0001). CONCLUSIONS: AFP-driven RR production by hepatoma cells significantly enhances herpes viral cytotoxicity and specificity in vitro and reduces tumor burden in vivo.     

A novel bioluminescent tumor model of human renal cancer cell lines: an in vitro and in vivo characterization

PURPOSE: Bioluminescent imaging permits sensitive in vivo detection and quantification of cells engineered to emit light. We developed a bioluminescent human renal cancer cell line for in vitro and in vivo studies. MATERIAL AND METHODS: The 2 human renal cell carcinoma cell lines SN12-C and SN12-L1 were stably transfected to constitutively express luciferase using a retroviral shuttle. The bioluminescent signal was correlated with tumor cell numbers in vitro. Parental and transfected cells were compared by growth kinetics and histology. Tumor burden after heterotopic injection in immune deficient mice was monitored up to 39 days. The kinetics of the bioluminescent signal was evaluated for 1 to 60 minutes following luciferin injection. RESULTS: Bioengineered renal cancer cell lines stably expressed luciferase. The growth kinetics of the cells in vitro and the histology of tumors resulting from implantation of these cells were unaffected by retroviral transfection with the luciferase gene. As few as 1,000 cells could be reliably detected. The intensity of the bioluminescent signal correlated with the number of tumor cells in vitro. Photon emission in vivo and ex vivo correlated significantly with tumor weight at sacrifice. After intraperitoneal injection of luciferin there was a time dependent change in the intensity of the bioluminescent signal with maximum photon emission at 20 minutes (optimal 17 to 25). CONCLUSIONS: Luciferase transfected human renal cancer lines allow reliable, rapid, noninvasive and longitudinal monitoring of tumor growth in vivo. The ability to assess tumor development in vivo with time is economical and effective compared to end point data experiments.     

长链非编码RNA RP11-363G15.2通过抑制锌指蛋白8基因的表达对肾癌细胞迁移和增殖的影响

目的探讨长链非编码RNA-RP11-363G15.2在肾癌中的表达及对肾癌细胞PHF8基因表达的抑制作用。方法实时定量荧光聚合酶链反应(qRT-PCR)检测12对肾癌组织及癌旁组织、肾癌细胞株(ACHN、OS-RC-2、786-O、A498)及人近端肾小管细胞HK-2中RP11-363G15.2的表达。选取表达水平最低的肾癌细胞,分别转染RP11-363G15.2质粒(实验组)和阴性对照质粒(对照组)。qRT-PCR检测两组细胞中RP11-363G15.2和锌指蛋白8(PHF8)mRNA的表达。Western blot检测两组细胞中PHF8、细胞周期蛋白D1(Cyclin D1)、细胞周期依赖性激酶6(CDK6)、波形蛋白(Vimentin)和神经-钙粘素(N-cadherin)蛋白的表达。Transwell迁移实验和四甲基偶氮唑蓝(MTT)法分别检测两组细胞的迁移能力和增殖能力。结果qRT-PCR结果提示,肾癌组织中RP11-363G15.2的表达明显低于癌旁组织(P<0.01),肾癌细胞株中RP11-363G15.2的表达均明显低于人近端肾小管细胞(P<0.01),OS-RC-2细胞中RP11-363G15.2的表达最低(P<0.01)。与对照组相比,实验组OS-RC-2细胞中RP11-363G15.2表达明显增加[(1.06±0.19)vs.(11.09±1.48),t=6.75,P<0.01],PHF8 mRNA表达明显降低[(1.01±0.09)vs.(0.31±0.05),t=6.66,P<0.01]。Western blot结果提示PHF8蛋白表达减少,CDK6、Cyclin D1、Vimentin和N-cadherin蛋白表达减少。与对照组相比,转染RP11-363G15.2后的OS-RC-2细胞迁移能力降低(P<0.01),从第3天开始细胞增殖能力明显降低(P<0.01)。结论RP11-363G15.2在肾癌中低表达,RP11-363G15.2可通过下调肾癌细胞OS-RC-2中PHF8基因的表达,抑制细胞的迁移能力和增殖能力,可能为肾癌的治疗提供了一个新的分子靶点。     

Novel HER2 selective tyrosine kinase inhibitor, TAK-165, inhibits bladder, kidney and androgen-independent prostate cancer in vitro and in vivo

PURPOSE: TAK-165 is a new potent inhibitor of human epidermal growth factor receptor 2 (HER2) tyrosine kinase. Several reports suggest HER2 expression in bladder cancer, renal cell carcinoma (RCC) and androgen-independent prostate cancer. We therefore investigated the antitumor effect of TAK-165 on these urological cancer cells. MATERIALS AND METHODS: Western blot analysis was performed to confirm HER2 expression in cell lines. To study in vitro efficacy, cells were treated with TAK-165 at various concentrations for 72 h and then counted using a hemocytometer. Then the IC50 value was calculated. In the xenograft model, after the tumor reached 200-300 mm3 in volume, mice were orally administered TAK-165 10 mg/kg per day or 20 mg/kg per day or saline for 14 consecutive days (n=6-8). RESULTS: HER2 expression was observed in HT1376, UMUC3, T24 (bladder), ACHN (kidney), DU145, LNCaP, LN-REC4 (prostate), although the expression level in these cells was weak compared with BT474 (a breast cancer cell line which expresses HER2 strongly). IC50 was varied from 0.09 to greater than 25 micromol/L in the bladder cancer cell line. ACHN cells were less sensitive in vitro. The prostate cancer cell lines studied were all sensitive (IC50 0.053-4.62 micromol/L). In the xenograft model, treatment with TAK-165 significantly inhibited growth of UMUC-3, ACHN, and LN-REC4. The antitumor effect (T/C [%]=growth of TAK-165 treated tumor/average growth of control tumorx100) after 14 days treatment were 22.9%, 26.0%, and 26.5% in UMUC3, ACHN and LN-REC4, respectively. CONCLUSIONS: TAK-165 may be a hopeful new agent for bladder, kidney and androgen-independent prostate cancer.