William Hansel

Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo.

We have prepared conjugates of a membrane disrupting lytic peptide (hecate) and a 15-amino acid segment of the β-chain of CG and hecate and the decapeptide, luteinizing hormone releasing hormone (LHRH). We have tested the concept that these conjugates will target breast cancer cells expressing LH/CG or LHRH receptors. In previous studies, we were able to destroy prostate cancers in vitro and in vivo with lytic peptide conjugates [1]. Hecate, hecate–βCG and LHRH–hecate were added to cultures of the human breast cancer cell lines MCF-7 and MDA-MB-435S. Hecate and its conjugates showed concentration dependent toxicity to both cell lines. The lytic peptide alone showed similar EC50 values for both cell lines; however, there was a significant difference between the EC50 values when the conjugates were tested. The hormone dependent MCF-7 cell line was less sensitive to the βCG conjugate than to the LHRH conjugate; the reverse was found for the hormone independent MDA-MB-435S cells. Removal of steroids decreased the sensitivity of MCF-7 cells to both lytic peptide conjugates and this sensitivity could be restored by adding estradiol. Activation of protein kinase C further increased the sensitivity to the drug. MDA-MB-435S xenografts were established in intact female athymic nude mice, which were treated once a week for 3 weeks with hecate–βCG via the lateral tail vein. The ability of hecate–βCG to destroy xenografts of human breast cancer cells (MDA-MB-435S) in nude mice was demonstrated for the first time. We conclude that hecate–βCG and LHRH–hecate conjugates could serve as useful drugs for the treatment of breast cancer.

Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo

In a series of in vivo and in vitro experiments, the concept has been established that breast cancer cells that express LH/CG or LHRH receptors can be targeted and destroyed by constructs consisting of a lytic peptide moiety and a 15-amino acid segment of the beta-chain of CG or by an LHRH lytic peptide conjugate. Data obtained in vitro established the validity of this concept, showed the specificities of the Hecate-betaCG, and Phor14 and Phor21-betaCG conjugates in killing cells that express functional LH/CG receptors and proved that the LH/CG receptor capacity is directly related to the compounds specificity. In in vivo experiments, Hecate-betaCG, Phor14-betaCG, and Phor21-betaCG(ala) each caused highly significant reductions of tumor volume and tumor burden in nude mice bearing breast cancer xenografts; Hecate and Phor21 alone or conjugated with non-specific peptides were not effective. Most importantly, the lytic peptide conjugates were all highly effective in targeting and destroying disseminated breast cancer metastases in lymph nodes, bones, lungs and other organs.

Targeting Breast and Prostate Cancers Through Their Hormone Receptors

Abstract A targeted treatment that effectively destroys human breast, prostate, ovarian, and testicular cancer cells that express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors has been developed. The treatment consists of a conjugate of a membrane-disrupting lytic peptide (Hecate, Phor14, or Phor21) and a 15-amino acid segment of the beta chain of CG. Because these conjugates act primarily by destroying cell membranes, their effects are independent of cell proliferation. The conjugates are relatively small molecules, are rapidly metabolized, and are not antigenic. In a series of independent experiments conducted in three different laboratories, the validity of the concept has been established, and it has been shown that the LH/CG receptor capacity of the cancer cells is directly related to the sensitivity of the lytic peptide conjugates. Sensitivity to the drugs can be increased by pretreating prostate or breast cancer cells with FSH or estradiol to up-regulate LH/CG receptors. A series of 23 in vivo experiments involving a total of 1630 nude mice bearing xenografts of human prostate or breast cancer cells showed convincingly that all three lytic peptide-betaCG compounds were highly effective in destroying tumors and reducing tumor burden. Hecate-betaCG was less effective in mice bearing ovarian epithelial cancer cell xenografts, but was highly effective in treating granulosa cell tumors in transgenic mice. In addition, Hecate-betaCG and Phor14-betaCG were highly effective in targeting and destroying prostate and breast cancer cell metastases in the presence or absence of the primary tumors. Although effective in vitro, neither Hecate nor Phor14 alone were effective in reducing primary tumor volume or burden in nude mice bearing prostate or breast cancer xenografts.

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors

We have prepared a conjugate of a lytic peptide (hecate) and a 15‐amino acid segment of the β‐chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors.

Conjugates of lytic peptides and LHRH or βCG target and cause necrosis of prostate cancers and metastases

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

Pharmacokinetics and pharmacodynamics of Phor21-βCG(ala), a lytic peptide conjugate

Phor21‐βCG(ala), a 36‐amino acid peptide comprised of a lytic peptide (Phor21) conjugated to a modified 15‐amino acid segment of the β‐chain of chorionic gonadotropin (βCG(ala)), selectively kills cancer cells that over‐express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors by disrupting cellular membrane structure. These studies were designed to further characterize its in‐vitro inhibition and in‐vivo destruction of prostate cancer cells, biostability and pharmacokinetics to determine its pharmacokinetic and pharmacodynamic profile. Inhibitory effects of Phor21‐βCG(ala) were tested in PC‐3 and Caco‐2 cells as well as in nude mice bearing PC‐3 cells transfected with the luciferase gene (PC‐3.luc). Plasma stability, protease hydrolysis and pharmacokinetics of Phor21‐βCG(ala) were measured by using liquid chromatography mass spectrometry (LC/MS/MS). Phor21‐βCG(ala) selectively inhibited proliferation in‐vitro and in‐vivo metastases of PC‐3 cells. Phor21‐βCG(ala) was relatively stable in mouse, rat, dog and human plasma. Its degradation was partially due to protease hydrolysis and thermodynamic catalysis. Intravenous administration of Phor21‐βCG(ala) showed its blood Cmax and AUC0→∞ around the in‐vitro effective levels. In the tested rodents, Phor21‐βCG(ala) displayed a moderate volume of distribution at steady state (VdSS) and slow clearance (Cl) in the rodents. In conclusion, Phor21‐βCG(ala) displayed promising in‐vitro and in‐vivo anti‐cancer activity with favourable pharmacokinetics, and may offer a novel approach to metastatic cancer chemotherapy.

The Essentiality of the Epididymal Fat Pad for Spermatogenesis

In 1935, two landmark publications by Cornell University scientists established the dogma that longevity and reproductive performance in rodents are primarily controlled by energy intake. McCay, Crowell, and Maynard (1) reported that the lifespans of rats maintained on calorie-restricted diets that retarded growth were prolonged, especially in males. In the same year, Asdell and Crowell (2) showed that sexual development in these growth-retarded rats was also retarded; vaginal opening and age at first estrus were delayed, and in contrast to ad libitum-fed rats, the two events became completely dissociated. After World War II, these findings were extended to domestic animals, where it was shown, for example, that calves fed from birth to sexual maturity at 130% of the recommended total digestible nutrient (TDN) allowances first came into estrus at 37.4 wk of age, compared with 49.1 wk for calves fed 100% of recommended TDN allowances and 72.0 wk for calves fed 70% of recommended TDN allowances (3). Similar data were obtained for age at the onset of spermatogenesis in male calves. Greater longevity of dairy cattle fed the 70% recommended TDN allowance was demonstrated by 1964 (4), and later the ability of caloric restriction to increase longevity was demonstrated in species as diverse as flies (5), worms (6), yeast (7), rodents (8), and monkeys (9). Because the calorie-restricted and control diets used in these early studies contained equal amounts of fat, most workers assumed that any influence of fat on reproduction was through its contributions to energy metabolism. However, by 1963, evidence that fat plays an important role in reproductive performance was obtained by Kennedy and Mitra (10) and by Frisch and McArthur in 1974 (11). These studies were the first to indicate that adipose tissue plays a direct role in female reproduction and suggested the need for a minimal amountof fat fornormal reproductivedevelopment.Finally, the discovery of leptin, a peptide hormone produced only in fat cells, removed any doubt as to the ability of fat cells to act as an endocrine organ and firmly established the role of adipose tissue in reproductive performance (12). Now, in what may become another landmark paper, Chu et al. (13) have extended these early results by establishing, for the first time, that a specific fat depot, the epididymal fat pad, produces a locally acting factor responsible for maintaining spermatogenesis in the hamster. Removal of the epididymal fat pad interrupts spermatogenesis and increases FSH concentration but does not affect testosterone, LH concentration, or mating behavior. However, it increases serum FSH concentrations. Transplantation of the excised epididymal fat pad to a sc site did not restore spermatogenesis. Removal of comparable amounts of white adipose tissue from other sites (inguinal) was also without effect, disproving the idea that the effect is due to a decreased energy supply and/or the need for some minimal amount of fat. The authors do point out the possibility that altered testicular blood flow, resulting in increased testicular temperature, might be an alternative explanation for the inhibition of spermatogenesis that occurs after removal of the epididymal fat pad. However, they do not think this is the case, because of the histological normality of the testicular interstitial cells and the normal testosterone production. They also point out that denervation of the testes at both poles, including the pampiniform plexus, which is more likely to produce damage to the venous drainage than epididymal fat pad removal, did not affect spermatogenesis. If the authors are correct in these conclusions, the race is on to identify any compounds of epididymal fat pad

The effects of a low therapeutic dose of βCG fragment–lytic peptide conjugates on ovarian function and gonadotropin secretion in ewes: A randomized controlled trial

The main objective of this experiment was to determine and compare the effects of two lytic peptide conjugates, Phor21-ßCG(ala) and ßCG(ala)-Phor21, at a low therapeutic dose (0.2 mg/kg body weight i.v.), on periovulatory ovarian and endocrine activity, and ensuing luteal function in an ovine experimental model. We hypothesized that the dense expression of LH/hCG receptors on the preovulatory follicle would present an appropriate target for the drugs and disrupt normal ovarian dynamics in sheep. Serum levels of reproductive hormones and ultrasonographic images were used for the assessment of periovulatory events following drug administration in 14 Rideau Arcott ewes; seven animals served as controls. Ovulations were synchronized with intravaginal progestogen-releasing sponges (medroxyprogesterone acetate, 60 mg) that were left in place for 12 days and a single i.m. injection of 750 IU of equine chorionic gonadotropin (eCG) given at sponge withdrawal. Both drugs were administered by i.v. injection 36 h post sponge removal/eCG injection, during the period of increasing LH responsiveness of potential ovulatory follicles and around the expected onset of the preovulatory surge of gonadotropins. No difference (p>0.05) was detected in the number of luteal structures per ewe in control versus treated animals during early luteogenesis. After drug administration, peak FSH concentrations were higher (p<0.05) in Phor21-ßCG(ala)-treated compared to control ewes and circulating estradiol concentrations were lower (p<0.05) in ßCG(ala)-Phor21-treated animals. Mean serum progesterone concentrations were lower (p<0.05) in ßCG(ala)-Phor21-treated than control ewes during the luteal phase post-treatment. There were no differences (p>0.05) in the percentage of ewes that lambed or lamb characteristics between the three groups at lambing 9 months post-treatment. In summary, neither Phor21-ßCG(ala) nor ßCG(ala)-Phor21 demonstrated adverse effects on the ovulatory process but the treatment with ßCG(ala)-Phor21 significantly depressed follicular and luteal steroidogenesis. With a lack of evidence for disruptive effects on endocrine function and fertility, these obsevations support the use of Phor21-ßGG(ala) as a cancer pharmaceutical.

Abstract 4128: Anti-angiogenic and anti-metastatic activities of juglone in pancreatic cancer cells

Pancreatic cancer is the fourth leading cause of cancer related deaths in the US. The late diagnosis, early metastasis and poor survival rate make this disease lethal. This current study was designed to examine the effect of juglone, 5 hydroxy-1, 4-naphthoquinone in modulating the angiogenic and metastatic potential of pancreatic cancer cells in vitro, using the MIA Paca-2 human pancreatic cancer cell line. Juglone is a natural quinoid abundantly found abundantly in plants of Juglandacea family. To understand the anti-angiogenic and anti-metastatic properties of juglone, expression of markers related to angiogenesis were measured by Western blot or ELISA and the effect on ability of cancer cells to migrate and invade after treatment was analyzed by transwell invasion and migration assay, wound healing assay, and in vitro tube formation assay in human umbilical vein endothelial cells. The Akt pathway is highly upregulated and is responsible for the regulation of various biological processes in pancreatic cancer cells including proliferation, growth, survival and angiogenesis. Akt activation is also associated with regulation of key angiogenic markers such as, VEGF (Vascular Endothelial Growth Factor) and HIF-1 (Hypoxia Inducible Factor). Upon treatment of pancreatic cancer cells with juglone, reduced expression of serine-phosphorylated Akt expression was observed. Juglone inhibited metastasis of pancreatic cancer cells as indicated by the results of cell migration, invasion and wound healing assay. Juglone treatment also downregulated the levels of VEGF and HIF-1α. Significantly, it was found that juglone inhibited pancreatic cancer cell motility, migratory and invading capabilities. These findings suggest that Akt inhibitors such as juglone possess ability to attenuate the aggressiveness of pancreatic cancer cells and can be used as a novel anti-cancer agent to prevent metastasis of highly invasive pancreatic adenocarcinomas. Citation Format: Namrata Karki, Frank Greenway, William Hansel, Sita Aggarwal, Jack N. Losso. Anti-angiogenic and anti-metastatic activities of juglone in pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4128.

Abstract B56: Inactivation of LKB1-AMPK pathway is mediated by inflammation in pancreatic cancer

Goal: The goal of this project is to determine if there is link between cancer cell metabolism and inflammation. LKB1is a known serine/threonine kinase tumor suppressor gene and it functions by sensing changes in cellular energy homeostasis and modulates anabolic and catabolic processes by activating its downstream kinase, AMP-activated protein kinase (AMPK). It is associated with Peutz-Jeghers syndrome, as well as various cancers including pancreatic cancer. Thus; it is a unique link between cell metabolism and inflammation. Hypothesis: Our hypothesis is that LKB1-AMK succumbs to inactivation processes because of increased NF-κB expression in pancreatic cancer cells and is associated with increased pancreatic cancer growth. Results: We tested five pancreatic cancer cells that express constitutive active NF-κB (pp65) and LKB1proteins by western blot analysis. In these cancer cells there was loss of active AMPK, phospho AMK (pAMPK) protein and its substrate pACC protein, suggesting a negative correlation between inflammation and loss of active AMPK. To strengthen this observation, we treated macrophage with LPS/IFNγ to create inflammatory environment and examined the protein levels of pp65, NF-κB regulated inflammatory gene (COX-2), pAMPK and pACC. We found that induction of pp65 and COX-2 are negatively correlated with pAMPK and pACC. To further support this observation we treated the pancreatic cancer cells with Metformin, a known activator of AMPK and our results clearly indicates that Metformin inhibits the proliferation of pancreatic cancer cells in a time-dependent manner. Metformin activates AMPK and ACC in pancreatic cancer cells. These data indicate that, compounds that activates LKB1-AMPK pathway can be beneficial in pancreatic cancer treatment. Citation Format: Sita Aggarwal, Qingxia Wang, Shailendra Giri, William Hansel. Inactivation of LKB1-AMPK pathway is mediated by inflammation in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B56.