Adenoviral vector containing wild-type p16 suppresses prostate cancer growth and prolongs survival by inducing cell senescence

It is estimated that there will be >184,500 new cases of prostate cancer and 42,000 prostate cancer deaths in the United States this year. In the majority of patients diagnosed with prostate cancer, the disease will be too advanced for cure with standard medical treatment. New therapeutic strategies against advanced prostate cancer are desperately needed. As alterations in tumor-suppressor gene p16 are common in prostate cancer, one novel approach is gene therapy using a replication-deficient, E1/E3-deleted adenovirus type 5 containing a p16 under the control of a truncated Rous sarcoma virus promoter (AdRSVp16). In vitro, PC-3 cells that had been stably transfected with p16 expression vector under the control of an inducible promoter had a 70% reduction in cell number compared with the parental and control vector-transfected PC-3 cells. Similarly, AdRSVp16 significantly inhibited the growth of PPC-1 and PC-3 prostate cancer cells in culture. Furthermore, PPC-1 tumors grown in nude mice treated by a single injection of AdRSVp16 had a marked reduction in tumor size compared with untreated control-treated or viral control-treated PPC-1 tumors. Animals bearing tumors treated with AdRSVp16 also had longer survival. Adenovirally mediated expression of transgene was detected in xenograft tumors for at least 2 weeks. Taken together, these results suggest that AdRSVp16 should be considered for prostate cancer gene therapy in human clinical trials.     

Intraperitoneal delivery of hrR3 and ganciclovir prolongs survival in mice with disseminated pancreatic cancer

BACKGROUND AND OBJECTIVES; Intraperitoneal dissemination of pancreatic cancer is associated with a poor prognosis. Surgical resection does not prolong survival. Here we describe a novel approach to this difficult clinical problem consisting of intraperitoneal delivery of the herpes simplex virus (HSV) vector (hrR3) to mice with peritoneal dissemination of the pancreatic cancer cells. METHODS: The human pancreatic cancer cell line (SW1990) was implanted into the abdominal cavity of nude mice. Fifteen days later, the abdominal neoplasm was treated by intraperitoneal injection of the replication-conditional HSV vector (hrR3). The mutant lacks the ribonucleotide reductase gene, but contains an intact HSV-tk gene. Beginning 5 days after vector injection, mice were treated with a 14-day course of ganciclovir. RESULTS: Long-term survival (150 days) was seen in 70% of mice receiving hrR3 and ganciclovir, 40% of mice receiving hrR3 alone, and 0% of untreated mice. No vector-related mortality was observed. X-Gal tissue staining revealed blue-stained cells only in tumor nodules, not in normal organs. CONCLUSIONS: Intraperitoneal delivery of hrR3 and ganciclovir improves survival in this murine model of peritoneal dissemination of pancreatic cancer. The ability of hrR3 to replicate only in rapidly dividing cells makes this virus an attractive vector for gene therapy of cancer.     

Zoledronic acid suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing mice

BACKGROUND: Although there is no doubt that bisphosphonates (BPs), specific inhibitors of osteoclasts, are beneficial for the treatment of bone metastases, their effects on visceral metastases are unclear. The effect of zoledronic acid (ZOL) was examined in vivo on lung metastasis progression and animal survival, and in vitro on the cellular mechanisms involved. METHODS: An animal model of lung metastasis was developed in C3H/He mice inoculated intravenously with a spontaneous murine osteosarcoma POS-1 cell line. Lung metastasis was determined at the time of autopsy. ZOL was assessed in vitro on POS-1 cell proliferation, cell cycle progression, and caspase-1 and -3 activities. RESULTS: The overall survival in five independent experiments (two series treated with ZOL 0.1 mg/kg twice a week, and three series with 0.1 mg/kg five times a week) showed a significant increase of the actuarial survival: 0.422 +/- 0.07 in ZOL-treated animals versus 0.167 +/- 0.07 in controls (P = 0.036). Lung metastases were absent in all ZOL-treated mice that survived more than 21 days postinjection as revealed by macroscopic and histologic analysis. In vitro, a 48-hour incubation with 10 microM ZOL inhibited POS-1 cell line proliferation associated with cell cycle arrest in S-phase. In addition, ZOL induced a weak increase of caspase-3 activity, but not caspase-1. CONCLUSION: We demonstrate that ZOL exerts a direct antitumor effect on POS-1 cells in vitro, significantly diminishes osteosarcoma-induced lung metastasis in vivo, thereby prolonging survival of POS-1-inoculated animals.     

Peritumoral injection of adenovirus vector expressing NK4 combined with gemcitabine treatment suppresses growth and metastasis of human pancreatic cancer cells implanted orthotopically in nude mice and prolongs survival

NK4 or adenovirus vector expressing NK4 (Ad-NK4) can act bifunctionally as a hepatocyte growth factor antagonist and angiogenesis inhibitor and has potential value in cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of Ad-NK4 in combination with gemcitabine (GEM) against pancreatic cancer. In vitro study showed a strong antiproliferative effect of GEM and a potent anti-invasive effect of Ad-NK4 against pancreatic cancer cells. In in vivo experiments, SUIT-2 human pancreatic cancer cells were implanted into the pancreas of nude mice. Mice were treated with Ad-NK4 by injection into the peritumoral region of the pancreas on day 5 after implantation followed by weekly i.p. injections of GEM. On day 28 after implantation, pancreatic tumor volume was significantly smaller than that in mice treated with Ad-LacZ, Ad-NK4 alone, or GEM alone. Furthermore, combination therapy completely suppressed peritoneal dissemination and liver metastases, leading to significantly increased survival. Histologic and immunohistochemical assays of primary tumors indicated that combination therapy prohibited both cell proliferation and angiogenesis, resulting in high levels of apoptosis. These results suggest that peritumoral injection of Ad-NK4 plus GEM is a potent combination therapy for pancreatic cancer.     

Epidermal growth factor prolongs survival time of tumor-bearing mice

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.     

Rottlerin suppresses growth of human pancreatic tumors in nude mice,and pancreatic cancer cells isolated from Kras mice

The purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growth of human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from Kras^G12D mice. AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by the immunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreatic cancer cells isolated from Kras^G12D mice were also examined. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial–mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras^G12D mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.     

CRBP suppresses breast cancer cell survival and anchorage-independent growth.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.     

Inhibition of group 1 p21‐activated kinases suppresses pancreatic stellate cell activation and increases survival of mice with pancreatic cancer

Pancreatic cancer remains one of the most lethal of all solid tumors. Pancreatic stellate cells (PSCs) are primarily responsible for the fibrosis that constitutes the stroma and p21‐activated kinase 1 (PAK1) may have a role in signalling pathways involving PSCs. This study aimed to examine the role of PAK1 in PSCs and in the interaction of PSCs with pancreatic cancer cells. Human PSCs were isolated using the modified outgrowth method. The effect of inhibiting PAK1 with group 1 PAK inhibitor, FRAX597, on cell proliferation and apoptosis in vitro was measured by thymidine incorporation and annexin V assays, respectively. The effect of depleting host PAK1 on the survival of mice with pancreatic Pan02 cell tumors was evaluated using PAK1 knockout (KO) mice. PAK1 was expressed in isolated PSCs. FRAX597 reduced the activation of PSCs, inhibited PSC proliferation, and increased PSC apoptosis at least in partial by inhibiting PAK1 activity. The decreased expression and activity of PAK1 in PAK1 KO mice tumors was associated with an increased mouse survival. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis. Targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.     

Adenovirus-mediated p16INK4 gene transfer significantly suppresses human breast cancer growth

The p16INK4 tumor suppressor gene encodes a protein that inhibits cyclin-dependent kinase 4, and its homologous deletion is common in human breast cancer. p16INK4 gene transfer has been reported to be efficacious in inducing growth inhibition of various human tumors such as brain, lung, prostate, and esophageal cancers. However, the efficiency of the p16INK4 gene with regard to growth inhibition of human breast cancer has not been studied extensively. To examine its tumor-suppressive function and its potential in breast cancer gene therapy, the wild-type p16INK4 gene was expressed in an adenovirus-mediated gene delivery system and introduced into breast cancer cell lines that do not express p16INK4 protein. Expression of the introduced p16INK4 blocked tumor cell entry into the S phase of the cell cycle, induced tumor cell apoptosis, and inhibited tumor cell proliferation both in vitro and in vivo. These results strongly suggest that p16INK4 is a tumor suppressor gene and suggest that it has potential utility in breast cancer gene therapy.     

联合铂类化疗改善晚期胰腺癌预后

目的探讨治疗晚期胰腺癌的有效药物。方法选择病理诊断明确的Ⅲ～Ⅳ期胰腺癌患者,至少接受2周期以上吉西他滨、氟尿嘧啶及铂类化疗,电话回访生存期,分析不同药物治疗后生存期变化。结果 95例晚期胰腺癌患者中位生存期7.1月,1年生存率30.5%,2年生存率8.4%,3年生存率3.2%。Cox单因素生存模型分析显示,Ⅳ期患者较Ⅲ期患者生存期短（P=0.002,HR：2.09,95%CI：1.31～3.35）,联合铂类化疗方案治疗患者较未联合患者生存期长（P=0.011,HR：0.56,95%CI：0.35～0.88）,胰体尾癌较胰头颈部癌患者生存期有缩短趋势,但差异无统计学意义（P=0.062,HR：1.53,95%CI：0.98～2.38）。Cox多因素生存模型分析显示,Ⅳ期患者较Ⅲ期患者死亡风险增加（P=0.026,HR：1.83,95%CI：1.08～3.10）,联合铂类化疗能降低晚期胰腺癌死亡风险（P=0.026,HR：0.59,95%CI：0.38～0.94）。结论Ⅳ期胰腺癌患者较Ⅲ期患者预后差,联合铂类化疗可能延长晚期胰腺癌生存期。     

p16基因抑制食管癌细胞生长的研究

目的 探讨ｐ１６基因对食管癌细胞生长的抑制作用,为食管癌致癌机理的研究和基因治疗提供理论基础。方法 我们采用基因转染技术,将全长野生型ｐ１６ ｃＤＮＡ转染到经亚硝胺诱发的人胎儿食管癌细胞系中,该细胞内源性ｐ１６基因已发生纯合性缺失。结果 转染ｐ１６基因的食管癌细胞生长受到抑制,其在软琼脂上克隆形成能力降低了大约４倍。对细胞周期分析表明,处在Ｇ０￣Ｇ１期的细胞由３３．７％增加到６８．６％。经Ｄｏｔ     

Adrenomedullin antagonist suppresses in vivo growth of human pancreatic cancer cells in SCID mice by suppressing angiogenesis

Since it is reported that adrenomedullin (AM) upregulated by hypoxia inhibits hypoxic cell death, we examined the effects of AM antagonist (AM C-terminal fragment; AM(22-52)) on the growth of pancreatic cancer cells. We, for the first time, demonstrated that AM antagonist significantly reduced the in vivo growth of the pancreatic cancer cell line. Immunohistochemical analysis demonstrated that the mean diameter of blood vessels was significantly smaller in the tumor tissues treated with AM antagonist than in those treated with AM N-terminal fragment (AM(1-25)), and that the PCNA-labeling index was lower in the former than in the latter. Then we demonstrated that AM antagonist showed no effect on the in vitro growth of the pancreatic cancer cell line. These results showed that AM played an important role in the growth of pancreatic cancer cells in vivo, suggesting that AM antagonist might be a useful tool for treating pancreatic cancers.     

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM‐M3 cells cultured in the presence and absence of β‐hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase‐tagged syngeneic VM‐M3 cells. Mice were fed a standard diet supplemented with either 1,3‐butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM‐M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM‐M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.     

Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic cancer cell lines.

PURPOSE: Vascular endothelial growth factor A (VEGF-A) is a potent angiogenic agent that binds to two high affinity VEGF receptors (VEGFRs), a process facilitated by the low affinity neuropilin receptors. Although VEGF-A is overexpressed in pancreatic ductal adenocarcinoma, it is not known whether the in vivo growth of multiple pancreatic cancer cells can be efficiently blocked by VEGF-A sequestration. EXPERIMENTAL DESIGN: Four human pancreatic cancer cell lines were grown s.c. in athymic nude mice. One cell line also was used to generate an orthotopic model of metastatic pancreatic cancer. The consequences of VEGF-A sequestration on tumor growth and metastasis were examined by injecting the mice with a soluble VEGFR chimer (VEGF-Trap) that binds VEGF-A with high affinity. RESULTS: VEGF-Trap, initiated 2 days after tumor cell inoculation, suppressed the s.c. growth of four pancreatic cancer cell lines and markedly decreased tumor microvessel density. Analysis of RNA from tumors generated with T3M4 cells revealed that VEGF-Trap decreased the expression of VEGFR-1 and neuropilin-1 and -2. VEGF-Trap, initiated 3 weeks after tumor implantation, also attenuated intrapancreatic tumor growth and metastasis in an orthotopic model using PANC-1 cells. CONCLUSIONS: VEGF-Trap is a potent suppressor of pancreatic tumor growth and metastasis and also may act to attenuate neuropilin-1 and -2 and VEGFR-1 expression. Therefore, VEGF-Trap may represent an exceedingly useful therapeutic modality for pancreatic ductal adenocarcinoma.     

Flt3 ligand delivered in a pluronic formulation prolongs the survival of mice with orthotopic pancreatic adenocarcinoma

Pancreatic adenocarcinoma is a devastating disease, characterized by asymptomatic development and extremely poor prognosis. Given the resistance of pancreatic cancer to standard chemo- and radiotherapy, we have focused on the development of immunotherapies for this disease. The number of dendritic cells (DCs), natural killer (NK) cells, and T-cells in the blood and secondary lymphoid organs is regulated by a group of hematopoietic growth factors, which includes fms-like tyrosine kinase-3 ligand (Flt3L). We have demonstrated previously that the bioavailability and in vivo half-life of Flt3L are increased by Flt3L formulation in the pluronic ProGelzx. In this study, we first examined the effectiveness of Flt3L delivered in ProGelz against subcutaneous (s.c.) pancreatic adenocarcinomas in mice. We found that an intramuscular (i.m.) injection of Flt3L in ProGelz significantly increased the survival of mice bearing s.c. pancreatic tumors, compared to the administration of phosphate-buffered saline (PBS) in ProGelz. We then tested Flt3L in ProGelz in an orthotopic pancreatic tumor model, and demonstrated that it significantly enhanced the survival of tumor-bearing mice, compared to PBS in ProGelz. Overall, these observations suggest that Flt3L formulated in ProGelz may have potential clinical utility as a treatment for pancreatic cancer.     

UVC irradiation suppresses platelet-derived growth factor-BB-induced migration in human pancreatic cancer cells

We have recently reported that short wavelength ultraviolet-C (UVC) irradiation inhibits cell growth and induces apoptosis in human pancreatic cancer cells. In this study, we investigated the effect of UVC on platelet-derived growth factor (PDGF)-BB-induced migration in pancreatic cancer cells, AsPC1 and BxPC3. In cell migration assays using a Boyden chamber Transwell, PDGF-BB exerted a maximum effect on migration of these cells at a dose of 70 ng/ml after 36 h of treatment. PDGF-BB also caused phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK) and Akt, but not of p38 MAPK in these cells. Pretreatment of these cells with UVC at a dose over 10 J markedly suppressed PDGF-BB-induced migration. Since UVC significantly inhibited PDGF-BB-induced phosphorylation of Akt, and subsequent glycogen synthase kinase (GSK) 3β, but not p44/p42 MAPK and SAPK/JNK, it is likely that UVC inhibits PDGF-BB-induced migration by suppressing the Akt-GSK3β pathway in pancreatic cancer cells. Taken together with our previous findings, UVC could be a useful tool for the treatment of patients with pancreatic cancer.     

S100P promotes pancreatic cancer growth, survival, and invasion.

PURPOSE: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. EXPERIMENTAL DESIGN: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. RESULTS: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. CONCLUSIONS: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.