Rafael Madero-Visbal

Small molecule tolfenamic acid inhibits PC‐3 cell proliferation and invasion in vitro, and tumor growth in orthotopic mouse model for prostate cancer

Specificity protein (Sp) transcription factors are implicated in critical cellular and molecular processes associated with cancer that impact tumor growth and metastasis. The non‐steroidal anti‐inflammatory drug, tolfenamic acid (TA) is known to inhibit Sp proteins in some human cancer cells and laboratory animal models. We evaluated the anti‐cancer activity of TA using in vitro and in vivo models for prostate cancer.

Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer

BACKGROUND AND OBJECTIVES To determine whether bioluminescence imaging of human lung cancer cells growing in an orthotopic murine model provides a sensitive tool for monitoring tumor progression in athymic nude mice. METHODS Human lung cancer (A549) cells were stably transfected with the firefly luciferase gene and inoculated into the right lung of athymic nude mice. Seven days after inoculation tumor growth was evaluated using the Kodak in-vivo Imaging System FX and continued to be monitored on a weekly basis. RESULTS In duplicate experiments, human lung cancer tumors formed in 90% of animals injected orthotopically. The mean intensity of the bioluminescence signal emitted from the lung cancer cells increased logarithmically during the course of study. Mice with positive bioluminescence signaling had confirmed tumors by microscopic histological analysis. Bioluminescence activity had a strong correlation with the tumor volume as determined histologically. CONCLUSIONS Bioluminescence intensity directly correlates with tumor volume and therefore offers a reliable approach for detecting and monitoring the growth of human lung cancer cells in orthotopic murine models.

Novel Murine Model for Colon Cancer: Non-Operative Trans-Anal Rectal Injection

BACKGROUND This study was conducted to develop a modified murine model of colon cancer that is non-operative. Currently, the most accurate orthotopic murine model of colon cancer requires an invasive procedure involving cecal injection of colon cancer cells and therefore limits the ability to perform immunological studies subsequent to cecal resections. MATERIALS AND METHODS Murine colon cancer (CT26) cells were injected submucosally into the distal, posterior rectum of BALB/c mice. Care was taken not to pass transmurally into the pelvic cavity. Different magnifications (10x versus 100x) were used for injection, and primary tumor growth and metastatic disease were studied. RESULTS In the initial study, 3/7 mice injected using 10x magnifications had notable, large tumor originating from the rectal wall, and histology revealed that all excised tumors were poorly differentiated adenocarcinoma. In the second study, 8/10 mice injected using 100x magnifications had notable tumor originating from the rectal well, and 4/8 mice had abnormal lung tissue with pathological evidence of hemorrhagic pulmonary edema. The use of 10x magnification resulted in 43% tumor take. In sharp contrast, 80% tumor take was observed with 100x magnification. The overall success of tumor take was 65% using the trans-anal rectal injection model. CONCLUSIONS Our modified orthotopic murine model of colon cancer offers an alternative non-operative murine model for colon cancer and is less invasive than the traditional orthotopic model (i.e., cecal injection). This model may allow for more accurate investigations of inflammation and immune responses to surgical intervention without the influence of previous abdominal surgery.

The Role of Parotidectomy in Sjögren's Syndrome

Sjögrens syndrome, a chronic and progressive autoimmune disorder mainly characterized by xerophthalmia, xerostomia, and parotid enlargement, is primarily managed medically, but some patients will require surgical management. Patients with Sjögrens syndrome have an increased risk of non-Hodgkin lymphoma. Superficial parotidectomy is indicated for diagnostic purposes and can be therapeutic in limited circumstances. Surgical indications for parotidectomy in Sjögrens syndrome include recurrent parotitis refractory to medical management; salivary gland malignancy; and severe, refractory pain. Surgical complications include transient or permanent facial nerve injury, post-operative pain, persistent inflammation of remnant parotid tissue, Frey syndrome, and facial scarring.

Abstract 619: O 6 -Benzylguanine inhibits tamoxifen-resistant breast cancer cell growth and resensitizes breast cancer cells to anti-estrogen therapy

Endocrine therapies using anti-estrogens are less toxic and very effective for breast cancers however tumor resistance to tamoxifen remains a stumbling block for successful therapy. Based on our recent study on the involvement of the DNA repair protein MGMT in pancreatic cancer (Clin Cancer Res. 15, 6087, 2009), we investigated whether MGMT overexpression mediates tamoxifen resistance. Specifically, we determined whether administration of MGMT inhibitor [O6-benzylguanine (BG)] at a non-toxic dose alone or in combination with the anti-estrogens (tamoxifen/fulvestrant) curtails human tamoxifen resistant breast cancer cell growth. Furthermore, we also determined whether BG sensitizes breast cancer cells to tamoxifen using tamoxifen resistant cells. MGMT expression was found to be increased in breast cancer cells relative to normal breast epithelial cells. Also, MGMT levels were significantly higher in tamoxifen resistant MCF-7 compared to the parent cells. Silencing of the ER-α expression using a specific siRNA resulted in augmentation of MGMT mRNA and protein levels by two fold. We also observed an inverse correlation between MGMT and p53 levels in breast cancer cell lines; moreover, p53 downregulation was accompanied by increased MGMT expression. Other experiments showed that BG alone or BG in combination with tamoxifen or fulvestrant decreased ER-α expression, whereas tamoxifen alone and fulvestrant alone increased and decreased the same respectively. All these treatments increased the p21cip1 mRNA and protein expression significantly. BG inhibited tamoxifen resistant breast cancer growth in a dose-dependent manner and it also resensitized resistant breast cancer cells to anti-estrogen therapy (TAM/ICI). These combinations also enhanced the cytochrome C release and PARP cleavage, indicative of apoptosis. In breast cancer xenografts, BG alone or a combination of BG with tamoxifen or fulvestrant caused a significant tumor growth delay and immunohistochemistry revealed that BG inhibited the expression of MGMT, ER- α, ki-67 and increased p21cip1 staining. These findings suggest that MGMT inhibition may provide a novel and effective approach for overcoming tamoxifen resistance (supported by a Florida Biomedical grant to SK). 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 619. doi:10.1158/1538-7445.AM2011-619

Abstract 1561: Guaiacol glyceryl ether, a novel agent to reduce MUC1 expression in breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: The overexpression and loss of polarity of Mucin1 (MUC1) has been documented in aggressive breast cancer. We have identified guaifenesin as a novel treatment to reduce or eliminate MUC1 expression on human breast cancer cells. Methods: MCF-7 and ZR-75-1 human breast cancer cells were grown for 48 hours in varying concentrations of guaifenesin to determine if a dose dependent effect on MUC1 protein expression resulted with treatment. In addition, cells were also harvested, RNA isolated, reverse transcribed and PCR was performed using ABI quantitative real time PCR machine. In another set of experiments, MCF-7 cells were plated and grown for 72 hours in the absence or presence of 12.5 mM guaifenesin and the cells were harvested and stained with propidium iodine for analysis by flow cytometry. The Cell-Titer Glo assay was performed on MCF-7 and ZR-75-1 cells incubated for 48 hours in the presence of guaifenesin at varying concentrations and the LD50 was determined. In a final set of experiments, MCF-7 cells, incubated in the presence or absence of guaifenesin, were analyzed for their ability to migrate /proliferate in vitro by using a standard scratch assay. For in vivo studies ovarectomized female nude mice were injected with MCF-7-luciferase tagged cells into the mammary fat pad 24 hours after an estrogen pellet was implanted into the right flank. Treatment (vehicle or 400 mg/kg/daily) was initiated after 10 days and tumor growth was monitored through Kodak imaging system. Six weeks after the treatment tumors were isolated, measured and processed for immunohistochemistry to analyze the expression of MUC1, TUNEL and CD31. Results: Initially, MUC1 gene expression was confirmed in MCF-7 and ZR-75-1 human breast cancer cell lines. Following confirmation, the LD50 was determined to be 12.5 mM. MUC1 gene expression was successfully decreased in a dose-dependent manner in both MCF-7 and ZR-75-1 cell lines when treated with guaifenesin. When MCF-7 cells are treated with 12.5mM guaifenesin, G1 arrest occurs within 24 hours and by 72 hours, 94.2% of cells are in G1 compared to 76.2% in control (drug free) conditions. In addition, there was a significant decrease in the ability of guaifenesin treated MCF-7 cells to migrate into the scratch compared to the control (untreated) cells. In vivo studies revealed that guaifenesin significantly decreased the breast tumor weight and volume which was associated with a low expression of MUC1 in tumor sections as evaluated by immunohistochemistry. Conclusion: These findings suggest that guaifenesin may provide a novel approach to inhibit breast cancer growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1561.

Abstract 4075: Tolfenamic Acid Inhibits Prostate Cancer Cells Growth and Tumor Development in Orthotopic Mice

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Transcription factors, Specificity proteins (Sp) play a critical role in growth and metastasis of cancer and there is clear evidence that Sp1 expression is a negative prognostic factor for survival in some cancer patients. Sp proteins regulate the expression of several genes, including PSA gene and c-Met which are associated with prostate cancer. Nonsteroidal anti-inflammatory drug, tolfenamic acid (TA) induces the degradation of specific Sp proteins (Sp1, Sp3 and Sp4) which play critical role in tumor growth and metastasis. Sp proteins also regulate the expression of survivin, a member of inhibitor of apoptosis gene family which is associated with resistance to chemotherapy and radiotherapy in several cancers. We have evaluated the anti-cancer activity of TA in prostate cancer. Methods and Results: Human prostate cancer cells (PC-3, DU-145 and LNcaP) were grown in the presence of DMSO (vehicle) or various concentrations (25, 50 and 100 µM) of TA and cell number and viability were measured at 24, 48, and 72 h post-treatment. Cell viability was measured using Cell Titr Glo kit (Promega) and the experiments were performed in triplicate. Cell lysates were prepared from the cells exposed to DMSO or 50 µM TA for 48 h and evaluated the expression of Sp proteins (Sp1, Sp3 and Sp4), and c-Met through Western blot analysis. For in vivo studies nude mice were injected with PC-3 Luc cells into the right prostate. Treatment (vehicle or 50 mg/kg TA/2 days) was initiated after one week and tumor growth was continuously monitored through Kodak imaging system. 4 weeks after the treatment tumors were isolated, measured and processed for immunohistochemistry to analyze the expression of Sp proteins and c-Met. In vitro studies show a significant decrease in the cell proliferation and viability and these results are dose and time dependent in all the cell lines. Furthermore, TA treatment for 48 h significantly decreased the expression of Sp1, Sp3 and c-Met in PC-3 cells. In vivo studies revealed that TA significantly decreased the tumor weight and volume which was associated with a low expression of Sp1, Sp3 and c-Met in tumor sections as evaluated by immunohistochemistry. Conclusion: Targeting key transcription factors such as Sp proteins using a small molecule compound, TA is a new strategy for the treatment of prostate cancer and our results are very crucial in the development of a safe, cost effective drug for the treatment of this devastating disease. Studies to test the effect of TA on critical genes associated with prostate cancer and TAs impact on developing higher response to radiation therapy are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4075.

Abstract 1403: Harnessing nanoparticles to improve toxicity after head and neck radiation

PURPOSE: To evaluate the ability of cerium oxide nanoparticles (CeO 2 ) to decrease xerostomia and skin reactions in athymic mice. METHODS: The head and neck (HN A) received no radiation exposure; B) received a single dose of 17.5 Gy; and C) received 30 Gy/6 fractions. In each cohort, animals were randomized into three groups (N=10 per group): 1) intraperitoneal (i.p.) injection of saline; 2) i.p. injection of 15 nM CeO 2; and 3) i.p. injection of 15 µM CeO 2 . Two independent double-blinded researchers graded radiation-induced dermatitis and hyperpigmentation at 1, 4, and 12 weeks after radiation therapy according to CTC v. 3.0 criteria. Ninety days after radiation, all mice were anesthetized and stimulated salivary flow was measured after subcutaneous pilocarpine injection (2mg/kg of B.W.) RESULTS: Stimulated sialometry strongly demonstrated improved salivary production in all CeO 2 groups compared to controls not receiving CeO 2 (mean salivary flow 204 vs. 115 µL/10min p=.0002). Grade 3 dermatitis was more prevalent in the fractionated vs. the single fraction cohort. In the fractionated cohort, the incidence of grade 3 dermatitis 1 week after radiation was decreased in the 15 µM CeO 2 group compared to the non-CeO 2 controls (10% vs. 100% incidence of Grade 3 dermatitis, respectively). A similar effect in reduction of grade 3 dermatitis was seen in the 15 µM CeO 2 group when compared to non-CeO 2 controls in both radiation cohorts for all time points evaluated. This effect was not appreciated in the 15 nM CeO 2 group. There was decrease in skin hyperpigmentation at 12 weeks in the 15 µM CeO 2 group compared to the 15 nM CeO 2 and non-CeO 2 groups (50, 70, and 90% grade 2, respectively). There were four Grade V toxicities in the fractionated cohort; three in the 15 nM CeO 2 group and one in the non-CeO 2 group. No Grade V toxicities were noted in the 15 µM CeO 2 group of mice. Additionally, there were no adverse effects noted in the groups of mice receiving CeO 2 without radiation. CONCLUSIONS: This study suggests that cerium oxide nanoparticles may have a radiation protective effect on salivary production. Parallel observations indicate a reduction in Grade 3 radiation-induced dermatitis and skin hyperpigmentation. The use of cerium oxide nanoparticles as a radioprotectant may be a feasible concept, but should be tested in a larger cohort of mice using a 15 µM concentration of CeO 2 . Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1403.