Susan Poulton

Establishment and Characterization of a New Human Prostatic Carcinoma Cell Line (DuPro-1)

A new human prostate adenocarcinoma cell line (DuPro-1) has been established from the athymic nude mouse supported xenograft DU5683. This was accomplished by embedding dispersed xenograft cells in 0.1 by 5.0 cm. spaghetti-like strands of Basement Membrane MATRIGEL [BMM (Collaborative Research, Inc.)], a unique technique facilitating the transition to tissue culture. Now passed over 30 times, the cells display anchorage and serum concentration independent growth with a doubling time of 22 to 24 hours. Cells exhibit pronounced morphological differences when grown on BMM coated culture dishes, assuming a pseudoglandular configuration, in contrast to typical homogeneous monolayer growth on plastic culture dishes. Light and electron microscopy show cohesive sheets of anaplastic epithelial cells, consistent with prostate carcinoma. Karyotypic analysis revealed all human chromosomes, near tetraploidy, 10 to 12 markers, and 3 to 4 X chromosomes, without a Y chromosome. Cells injected s.c. or embedded in BMM and implanted in the subrenal capsule space are equally tumorigenic in male and female athymic mice, suggesting that DuPro-1 cells are hormonally insensitive. Embedding cells in BMM may be useful in developing other tissue culture cell lines from neoplasms difficult to initiate in vitro. DuPro-1 should provide a valuable means to study the biology, immunology, and chemosensitivity of human prostate cancer.

Effect of intermittent fasting on prostate cancer tumor growth in a mouse model

Caloric restriction (CR) has been shown to have anti-cancer properties. However, CR may be difficult to apply in humans secondary to compliance and potentially deleterious effects. An alternative is intermittent CR, or in the extreme case intermittent fasting (IF). In a previous small pilot study, we found 2 days per week of IF with ad libitum feeding on the other days resulted in trends toward prolonged survival of mice bearing prostate cancer xenografts. We sought to confirm these findings in a larger study. A total of 100 (7- to 8-week-old) male severe combined immunodeficiency mice were injected subcutaneously with 1 × 105 LAPC-4 prostate cancer cells. Mice were randomized to either ad libitum Western Diet (44% carbohydrates, 40% fat and 16% protein) or ad libitum Western Diet with twice-weekly 24 h fasts (IF). Tumor volumes and mouse bodyweights were measured twice weekly. Mice were killed when tumor volumes reached 1000 mm3. Serum and tumor were collected for analysis of the insulin/insulin-like growth factor 1 (IGF-1) hormonal axis. Overall, there was no difference in mouse survival (P=0.37) or tumor volumes (P⩾0.10) between groups. Mouse body weights were similar between arms (P=0.84). IF mice had significantly higher serum IGF-1 levels and IGF-1/IGFBP-3 ratios at killing (P<0.001). However, no difference was observed in serum insulin, IGFBP-3 or tumor phospho-Akt levels (P⩾0.39). IF did not improve mouse survival nor did it delay prostate tumor growth. This may be secondary to metabolic adaptations to the 24 h fasting periods. Future studies are required to optimize CR for application in humans.

Effect of intermittent fasting with or without caloric restriction on prostate cancer growth and survival in SCID mice

Caloric restriction (CR) delays cancer growth in animals, though translation to humans is difficult. We hypothesized intermittent fasting (i.e., intermittent extreme CR), may be better tolerated and prolong survival of prostate cancer (CaP) bearing mice.

Low-Carbohydrate Diets and Prostate Cancer: How Low Is "Low Enough"?

Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm3. Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P < 0.04). Among the low-carbohydrate arms, NCKD-fed mice were significantly lighter than the 10% or 20% carbohydrate groups (P < 0.05). Tumors were significantly larger in the 10% carbohydrate group on days 52 and 59 (P < 0.05), but at no other point during the study. Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10–20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal). Cancer Prev Res; 3(9); 1124–31. ©2010 AACR.

The effect of carbohydrate restriction on prostate cancer tumor growth in a castrate mouse xenograft model

No‐ and low‐carbohydrate diets delay tumor growth compared to western diet (WD) in prostate cancer (PCa) xenograft studies. The effect of these diets in concert with androgen deprivation is unknown.

Dipyridamole-cisplatin potentiation: enhanced in vivo cytotoxicity in xenograft models of human testicular and bladder cancers.

The antitumor efficacy and host toxicity of dipyridamole (DP), methotrexate (MTX) and cisplatin (CDDP) alone and combined were evaluated in a nude mouse supported human bladder cancer model. Single agent post treatment tumor volume growth ratio [TGR] values of DP, MTX and CDDP were 97%, 65% and 49% of control. While the MTX/DP combination produced only mild cytotoxic enhancement, CDDP/DP and CDDP/MTX/DP reduced TGR to 20% and 17%, respectively. A second multi-dose evaluation of CDDP/DP using human testicular carcinoma in this model also showed a CDDP dose-dependent response with achievable complete tumor regression. Host toxicity was not substantially increased by DP. DP would appear to be effective in vivo as a chemosensitizer of CDDP; it may enhance the therapeutic efficacy of CDDP in a variety of tumors.

Effect of Isocaloric Low-Fat Diet on Prostate Cancer Xenograft Progression in a Hormone-Deprivation Model

PURPOSE Previous mouse studies suggesting that low fat diets slow prostate cancer growth often used corn oil (omega-6), which enhances prostate cancer growth, as the primary fat. Using a saturated fat based diet we previously found no significant difference in tumor growth between low and high fat fed SCID mice (Taconic Farms, Hudson, New York) xenografted with LAPC-4 cells. Whether similar results would hold in a castration model is unclear. MATERIALS AND METHODS A total of 80 male SCID mice were fed a Western diet (40% fat and 44% carbohydrate) and injected with LAPC-4 human prostate cancer cells. When tumors were 200 mm(3), the mice were castrated and randomized to an isocaloric Western or a low fat diet (12% fat and 72% carbohydrate). Animals were sacrificed when tumors were 1,000 mm(3). Serum was collected and assayed for prostate specific antigen, insulin, insulin-like growth factor 1 and insulin-like growth factor binding protein 3. Tumors were assayed for total and phosphorylated Akt. RESULTS Mouse weight was equivalent in the 2 groups. Overall dietary group was not significantly associated with survival (log rank p = 0.32). There were no statistically significant differences in prostate specific antigen (p = 0.53), insulin-like growth factor axis parameters (each p >0.05) or p-Akt-to-t-Akt ratios (p = 0.22) between the groups at sacrifice. CONCLUSIONS In this xenograft model we found no difference in tumor growth or survival between low fat vs Western fed mice when the fat source was saturated fat. These results conflict with those of other studies in which corn oil was used to show that low fat diets delay prostate cancer growth, suggesting that fat type may be as important as fat amount in the prostate cancer setting.

Increased biodiversity in the environment improves the humoral response of rats

Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of “natural” antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.

The therapeutic impact of dipyridamole: Chemopotentiation of the cytotoxic combination 5-fluorouracil/cisplatin in an animal model of human bladder cancer

Using an in vivo assay of tumor cytotoxicity (the subrenal capsule assay in nude mice), two therapeutic strategies for the treatment of advanced human transitional cell carcinoma have been evaluated: 1) the use of 5-fluorouracil in combination with cisplatin and 2) the ability of the chemosensitizer dipyridamole to augment the cytotoxicity of CDDP and 5FU. A moderate cytotoxic response of human TCC line DU-4284 to single agent CDDP was seen; it was dose dependent at minimally toxic doses [maximal cytotoxicity--27% tumor survival (%TS) relative to control]. Efficacy was further significantly enhanced by the addition of DP [11%TS (p = .008)]. 5FU at minimally toxic doses (100 and 150 mg./kg.) also demonstrated moderate dose-dependent cytotoxic activity (35 and 31%TS, respectively) which was further enhanced by DP [21%TS (p = .03) and 18%TS (p = .05)]. A constant dose ratio of CDDP/5FU when diluted showed dose-dependent cytotoxicity; at the highest dose dilution studied, substantial cytotoxic efficacy (17%TS) was attained. The cytotoxicity of 5FU/CDDP was order independent (p = .95). The addition of DP to this combination (5FU/CDDP) further enhanced efficacy; host toxicity was not substantially enhanced. A multiple regression analysis confirmed a statistically significant effect of DP with both CDDP and 5FU (p = .001 and 0.0001, respectively); tests for trend showed no significant interaction (p = 0.33 for all models). It is concluded that, in this preclinical in vivo model of human bladder cancer, 1) CDDP and 5FU show substantial enhanced efficacy when combined, and 2) DP serves as an in vivo chemosensitizer of both CDDP and 5FU; DP further augments the efficacy of this binary combination. These data would indicate the potential of this ternary (5FU/CDDP/DP) drug therapeutic regimen for clinical trial to treat advanced bladder cancer.

Enhanced in vivo cytotoxicity of recombinant human tumor necrosis factor with etoposide in human renal cell carcinoma

SummaryThe combination of tumor necrosis factor (TNF) and etoposide (ETP) was evaluated for potential cytotoxic efficacy against a human renal cell carcinoma xenograft using an in vivo assay employing an athymic mouse host with tumor implanted a the subrenal capsule site. Both antitumor efficacy (relative survival or RTS) and toxicity (weight loss) of TNF and ETP alone and in combination were evaluated. While TNF and ETP alone were mildly inhibitory (RTS 90% and 71%, respectively), the combination caused marked tumor inhibition (45% of controls). Host toxicity encountered with the combination did not exceed the toxicity associated with ETP alone, suggesting that the therapeutic index may have been augmented. It is concluded that enhanced antitumor activity without substantial augmentation of toxicity is observed with this combination, providing a rationale for further evaluation of tumor necrosis factor-based regimens for the treatment of advanced renal carcinoma.