Interacting Genetic Lesions of Melanoma in the Tumor Microenvironment: Defining a Viable Therapy.

Melanoma is the most aggressive form of skin cancer with an estimated 106,110 newly diagnosed cases in the United States of America in 2021 leading to an approximated 7180 melanoma-induced deaths. Cancer typically arises from an accumulation of somatic mutations and can be associated with mutagenic or carcinogenic exposure. A key characteristic of melanoma is the extensive somatic mutation rate of 16.8 mutations/Mb, which is largely attributed to UV exposure. Bearing the highest mutational load, many of them occur in key driver pathways, most commonly the BRAFV600E in the mitogen-activated protein kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA-approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK, which has greatly expanded therapeutic intervention following a melanoma diagnosis. Up until 2011, therapeutic options for metastatic melanoma were limited, and treatment typically fell under the spectrum of surgery, radiotherapy, and chemotherapy.Attributed to the extensive mutation rate, as well as having the highest number of neoepitopes, melanoma is deemed to be extremely immunogenic. However, despite this highly immunogenic nature, melanoma is notorious for inducing an immunosuppressive microenvironment which can be relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab, which target the molecules CTLA-4 and PD-1, respectively.A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are expressed not only by immune cells but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. The presence of immunomodulatory molecules in melanoma, using data from The Cancer Genome Atlas and validation of expression in two model systems, human melanoma tissues and patient-derived melanoma cells, revealed that the expression levels of B and T lymphocyte attenuator (BTLA), TIM1, and CD226, concurrently with the BRAFV600E mutation status, significantly dictated overall survival in melanoma patients. These molecules, along with herpesvirus entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues when compared to normal skin melanocytes and have unique roles to play in T cell activation. New links are being uncovered between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule, making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

Non-surgical and guided-bone-regeneration surgical management of Type-3b dens invaginatus with an apico-marginal defect: A case report.

BACKGROUND: Dens invaginatus (DI), an unusual developmental anomaly is a challenge for the operating dentist with regard to its diagnosis and treatment. This case report presents the successful management of a Type-3b DI in a permanent maxillary lateral incisor associated with a large radicular cyst and communicating apico-marginal defect (Von Arx type IIb). METHODS AND RESULTS: A 19-year-old female patient reported pain and palatal swelling. During the clinical examination, tooth #12 exhibited tenderness to percussion, and presented a deep periodontal pocket depth (PPD) of 12 mm, along with grade I mobility. Radiographic examination revealed a large peri-radicular radiolucency with atypical tooth morphology. Cone beam computed tomography clarified the complicated root canal anatomy to be Type-3b DI associated with an apico-marginal defect. The case was managed successfully by non-surgical endodontic therapy followed by surgical intervention utilizing a guided bone regenerative (GBR) approach. Eighteen-month follow-up showed an asymptomatic and functional tooth with a significant reduction in pocket depth. The periapical radiographs showed continued healing of the osseous defect. CONCLUSIONS: The successful healing outcome of a challenging case, characterized by a complex DI morphology, a large peri-radicular lesion, a through-and-through defect, and a combined endodontic-periodontal apico-marginal defect was achieved through accurate diagnosis, treatment planning, and execution using contemporary endodontic and periodontal treatment techniques. The application of GBR techniques during the surgical phase of treatment may have contributed to the improved regenerative healing outcome in this case, which was initially considered prognostically questionable. KEY POINTS: Why is this case new information? Type-3b DI exhibits a complex root canal structure, each case displaying unique characteristics, necessitating a case-specific treatment plan. In this case report the Type-3b DI morphology was associated with a large peri-radicular, through and through defect and combined endodontic periodontal apico-marginal defect. The treatment approach involved incorporating guided bone regenerative (GBR) principles during the surgical phase. This case report contributes to the existing evidence on the diagnosis and successful management of Type-3b DI with a concurrent apico-marginal defect. What are the keys to successful management of this case? The successful management of a prognostically challenging case was achieved through a closely integrated multidisciplinary coordination between the endodontist and periodontist. Utilization of contemporary techniques and tools contributed to the successful management The use of three-dimensional radiological examination through cone beam computed tomography enabled a precise preoperative assessment, facilitating the formulation of a treatment plan for managing both the Type-3b DI morphology and the associated peri-radicular lesion. Employing GBR techniques in peri-radicular surgery may have assisted in the healing of through-and-through periapical defects with concurrent apico-marginal defects (Von Arx type IIb). What are the primary limitations to the success of this case? A complex root canal anatomy associated with Type-3b DI morphology A large peri-radicular through and through defect with concurrent apico-marginal defect. Difficulty in weekly and long-term follow-up of the patient.

Eco-safe composite edible coating of hydrocolloids with papaya leaf extract improves postharvest quality and shelf life of papaya fruit under ambient storage.

Papaya postharvest management using low-temperature storage is discouraged as it is a tropical fruit. Extensive research is going on to preserve papaya quality at ambient storage using edible coatings and its composites. The present investigation examined the effects of an eco-safe composite edible coating consisting of hydrocolloid carboxymethyl cellulose (CMC) (1%), guar gum (1.5%), xanthan gum (0.3%), and Gum Arabic (10%) combined with papaya leaf extract (PLE) (1:1 ratio by volume) applied as dip treatment on "Red Lady" papaya fruit at ambient storage condition. Among all the attempted treatments, "PLE incorporated with CMC (1%)" was found to be the best, as the treated fruit exhibited the highest levels of biochemicals, whereas the lowest levels of physiological and enzymatic activity, which positively affected the shelf life. The "CMC + PLE" treatment enhanced the fruit gloss score by 70.1%, phenolics by 6.1%, ascorbic acid by 22.3%, total carotenoid content by 7.4%, and fruit predilection score by 22.0% over the control fruit. However, it lowered (controlling) the physiological loss in weight by 51.0%, decay incidence by 66.6%, and polygalacturonase and pectin methylesterase activity by 24.92% and 35.29%, respectively, over control. Moreover, this treatment exhibited the highest fruit purchase predilection score and prolonged the storage life for >3 days on the physiological loss standard basis (≤10%). This study indicates that "CMC (1%) with PLE (1:1)" composite coating application on papaya under ambient conditions might be an effective, environmentally friendly, and health-friendly way to retain the quality and extend the storage life.

A Novel Seed-Dressing Formulation Based on an Improved Mutant Strain of Trichoderma virens, and Its Field Evaluation.

Using gamma-ray-induced mutagenesis, we have developed a mutant (named G2) of Trichoderma virens that produced two- to three-fold excesses of secondary metabolites, including viridin, viridiol, and some yet-to-be identified compounds. Consequently, this mutant had improved antibiosis against the oomycete test pathogen Pythium aphanidermatum. A transcriptome analysis of the mutant vis-à-vis the wild-type strain showed upregulation of several secondary-metabolism-related genes. In addition, many genes predicted to be involved in mycoparasitism and plant interactions were also upregulated. We used tamarind seeds as a mass multiplication medium in solid-state fermentation and, using talcum powder as a carrier, developed a novel seed dressing formulation. A comparative evaluation of the wild type and the mutant in greenhouse under high disease pressure (using the test pathogen Sclerotium rolfsii) revealed superiority of the mutant over wild type in protecting chickpea (Cicer arietinum) seeds and seedlings from infection. We then undertook extensive field evaluation (replicated micro-plot trials, on-farm demonstration trials, and large-scale trials in farmers' fields) of our mutant-based formulation (named TrichoBARC) for management of collar rot (S. rolfsii) in chickpea and lentil (Lens culinaris) over multiple locations in India. In certain experiments, other available formulations were included for comparison. This formulation consistently, over multiple locations and years, improved seed germination, reduced seedling mortality, and improved plant growth and yield. We also noticed growth promotion, improved pod bearing, and early flowering (7-10 days) in TrichoBARC-treated chickpea and lentil plants under field conditions. In toxicological studies in animal models, this formulation exhibited no toxicity to mammals, birds, or fish.

Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine.

Transcription of several interferon-inducible human genes is also induced by double-stranded RNA. The nature and the mechanism of action of signals generated by interferons and by double-stranded RNA which mediate the induction of these genes are under investigation. Here we report that 2-aminopurine, a known inhibitor of protein kinases, could selectively block this induction process. Induction of mRNAs 561 and 6-16 in HeLaM cells by double-stranded RNA was completely inhibited by 10 mM 2-aminopurine, whereas cellular protein and RNA syntheses as well as the induction of metallothionein mRNA by CdCl2 were unaffected by this inhibitor. In addition, 2-aminopurine blocked the induction of the same two mRNAs and of mRNAs 2-5(A) synthetase, 2A, and 1-8 by alpha interferon and of mRNAs 2A and 1-8 by gamma interferon in HeLaM cells. The observed inhibition was at the level of transcription, and for establishing efficient inhibition, the 2-aminopurine treatment had to begin at early stages of interferon treatment. In GM2767 cells, 2-aminopurine inhibited induction of mRNAs 561 and 6-16 by double-stranded RNA but not by alpha interferon. These results suggest that double-stranded RNA-induced signal 2 is distinct from the interferon-alpha-induced signal 2 (R. K. Tiwari, J. Kusari, and G. C. Sen, EMBO J. 6:3373-3378, 1987) and that 2-aminopurine can block the former but not the latter. Moreover, it appeared that 2-aminopurine could block the production of signal 1 by interferons. This was confirmed by experiments in which we separately tested the effects of 2-aminopurine on signal 1 and signal 2 production by interferons in HeLaM cells. Although no direct experimental evidence is available as yet, our results are consistent with the hypothesis that the functioning of a protein kinase activity may be necessary for transcriptional induction of genes by double-stranded RNA and for gene induction by interferons in those cells in which signal 1 production is needed.

Pre-clinical toxicology and pathology of 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), a novel anti-cancer agent in male Beagle dogs.

We have developed a group of 4-substituted-1-nitroacridines with potent anti-tumor activity against prostate cancer and less toxic than parent 1-nitroacridines. The most active 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748) was selected for pre-clinical studies. The current study was undertaken to evaluate clinical and/or morphological adverse effects of C-1748 as a single intravenous dose at concentrations ranging from 0.16 to 4.6 mg/kg administered to male Beagle dogs. The maximum tolerated dose was 1.5 mg/kg. Emesis was observed in all groups lasting an average of 30 min to 12 h post-dosing. At high dose, extreme aggression was observed in one dog followed by disorientation and depression lasting for 48 h a frequent observation with chemotherapy. Reductions in platelets and white blood cells were observed which was similar to that seen with other chemotherapeutic agents. A compensatory hyperplasia of lymph nodes and a transient and limited extravasation in the intestinal mucosa were also observed. Increases in aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase were transient with normal levels restored by day 9. These enzyme increases were accompanied by epithelial hypertrophy of larger bile ductules in the periportal triads of the liver. The low toxicity profile and high tumor target activity make this novel class of drug a promising chemotherapeutic agent.

Selective responsiveness of human breast cancer cells to indole-3-carbinol, a chemopreventive agent.

BACKGROUND: Indole-3-carbinol (I3C), a compound found in cabbage, broccoli, and brussels sprouts, inhibits the growth of mammary tumors when fed to certain strains of mice. The chemopreventive and antitumor effects of I3C depend on the species and tissue type. The mechanism of action and specific human cell types that respond to I3C are not known. PURPOSE: Our purpose was to study the mechanism of action of I3C in estrogen-responsive (MCF-7) and estrogen-nonresponsive (MDA-MB-231) human breast cancer cell lines. METHODS: Estrogen responsiveness was determined by the ability of estradiol to stimulate the growth of breast cancer cells deprived of estrogen. The effect of I3C was measured on cell growth, estradiol metabolism, and level of cytochrome P-4501A1. Growth was measured by cell counts and soft-agar assay, estrogen metabolism was examined by a radiometric assay, and the level of cytochrome P-4501A1 was measured by Western blots with a polyclonal antibody. RESULTS: I3C inhibits the growth of estrogen-responsive cell line MCF-7 but has little effect on estrogen-nonresponsive cell line MDA-MB-231. Specific C-2 hydroxylation of estrogen and induction of cytochrome P-4501A1 was enhanced by I3C in the MCF-7 but not in the MDA-MB-231 cells. CONCLUSION: I3C has specific antigrowth effects in human breast cancer cells. The inhibitory effects of I3C may involve selective induction of estradiol metabolism and the related cytochrome P-450 system that may be limited to estrogen-sensitive cells.

Anti-carcinogenic and anti-metastatic properties of indole-3-carbinol in prostate cancer.

Indole-3-carbinol (I3C), a compound present as glucobracissin in cruciferous vegetables has anticancer activities which is in line with some of the epidemiological evidence that suggests a beneficial effect of consumption of cruciferous vegetables on cancer incidence and progression. The precise target of indole-3-carbinol has not been determined. We examined the effect of I3C on prostate cancer in a well-defined R3327 model using Copenhagen rats and the transplantable cell line, MAT-LyLu. This cell line derived from a tumor in Copenhagen rats is androgen independent and metastasizes to the lung and lymph nodes. Tumors were induced in Copenhagen rats by injecting MAT-LyLu subcutaneously and the animals treated with I3C that was administered either intraperitoneally or intravenously, in order to achieve maximal systemic exposure. This was a departure from the traditional chemopreventive route of indole-3-carbinol where the compound was incorporated in the diet. Our results indicate that I3C inhibited the incidence, growth and metastases of MAT-LyLu cells and both i.p. and i.v. injections of I3C were equally effective. Statistical analysis (Kaplan-Meier curves) clearly indicates a tumor-free and overall survival benefit as a result of treatment with I3C. These studies show for the first time that I3C in an injectible form has anti-prostate cancer activity.

Influence of genotype on the phospholipid fatty acid composition of splenic T and B lymphocytes in MRL/MpJ-lpr/lpr mice.

The MRL/MpJ-lpr/lpr mice manifest a T cell proliferative and autoimmune disorder. Similar changes occur much later in the life of MRL/MpJ-+/+ mice. MRL/MpJ-lpr/lpr (lpr/lpr) and MRL/MpJ-+/+ (+/+) mice were fed for six weeks nutritionally adequate semipurified diets containing 20% (w/w) fat, but differing in linoleic acid content. The phospholipid fatty acid composition of T and B cells was found to be dependent on genetic background of mice and level of linoleic acid in the diet. Changes in the levels of specific fatty acids like 16:0, 18:2 omega 6, 22:5 omega 3 and 22:6 omega 3 in some of the phospholipid components were observed in the MRL/MpJ-lpr/lpr strain in both the B and T cell types as compared with their normal +/+ counterpart strain. T cells of lpr/lpr mice exhibited significantly higher levels of 20:4 omega 6 than did T cells of other strain. High levels of dietary linoleic acid significantly increased incorporation of 18:2 omega 6 in T and B cells, while the effect on other fatty acids of the two types of cells varied with the phospholipid classes and fatty acids when compared with the low linoleic acid fed-group. Differences observed in the phospholipid fatty acid composition of the T and B cells of the congenic mice might contribute to differences in rate of progression of age-related changes suggesting that the autoimmune disorder might be mitigated by dietary manipulation.

A distinct kinase modulates the expression of IFN-inducible genes in human breast cancer cells.

The biological activity of interferons (IFNs) is presumed to be mediated through the induction of a number of IFN-inducible genes. IFN-mediated gene induction was examined in two human breast cancer cell lines, MCF-7 and BT-20. Both these cell lines were remarkably responsive to IFNs as a number of IFN inducible genes were rapidly induced. We examined the sensitivity of these genes towards 2-aminopurine (2-AP), a known inhibitor of double-stranded (ds) RNA dependent protein kinase. 2-AP has also been reported to inhibit the induction of IFN-beta 1 in response to dsRNA and the genes c-myc and c-fos in fibroblasts. In both MCF-7 and BT-20 cell lines, 2-AP selectively inhibited the IFN-induced gene responses. 2-AP did not affect levels of the oncogene, HER-2/neu. Tamoxifen (TAM), an antiestrogenic drug, which is known to inhibit the activity of protein kinase C at high concentrations, did not affect IFN-mediated gene induction. Our data is consistent with the concept that the 2-AP sensitive kinase is primarily associated with the IFN-induced gene systems and that positive and negative growth regulating stimuli in breast cancer may require the participation of distinct kinases.

Augmentation of cytotoxicity using combinations of interferons (types I and II), tumor necrosis factor-alpha, and tamoxifen in MCF-7 cells.

The cytotoxic effect of a combination of interferons (type I and II) and tumor necrosis factor (TNF), with an antiestrogenic drug, tamoxifen (TAM), was investigated in the estrogen receptor positive human breast carcinoma cell line, MCF-7. Cytotoxicity was measured by the MTT assay. In an attempt to define the molecular basis for the interaction between the interferons (IFNs) and TNF or any one of the cytokines with TAM, the induction characteristics of a number of IFN-induced mRNAs in response to IFNs, TNF, and TAM were studied. We observed an augmentation of the cytotoxic effect of TNF when it was combined with TAM. There appears to be an overlap in signalling mechanisms of IFNs and TNF as two of the IFN-inducible genes, 1-8 and 6-16 are also induced by TNF. mRNA 1-8 was induced by both IFN-alpha (type I) and IFN-gamma (type II). We conclude that TNF potentiates the cytotoxic effects of TAM in MCF-7 cells and that the three cytokines IFN-alpha, IFN-gamma, and TNF share some pathways that lead to specific induction of some cytokine responsive genes.

Induction of heat shock protein gp96 by immune cytokines.

Cytokines play a major role in regulating both humoral and cell-mediated immune responses. Recent advances in our understanding of cell-mediated immune responses have focused on the antigen presentation machinery and the proteins in the endoplasmic reticulum (ER). These proteins help the formation and stabilization of the major histocompatibility complex (MHC)-peptide interaction. A 96-kDa, ER-resident glycoprotein (gp96) is being evaluated as a therapeutic agent in cancer because of its ability to associate with a vast number of cellular peptides irrespective of size or sequence. Because the antigen presentation complex is assembled in the ER and a number of ER-resident proteins are modulated by cytokines, it is important to examine the regulation of gp96 in response to immune cytokines interferon gamma (IFN-gamma), and interleukin 2 (IL-2). Defects in signaling pathway in either of the cytokines can result in suboptimal immune response. We examined the effect of the cytokines IFN-gamma and IL-2 on the induction of gp96 in different cancer cell lines and examined the induction of DNA-binding proteins that recognize gamma interferon-activating sequence (GAS), present in the promoter region of gp96. The induction of GAS binding protein correlated with the induction of STAT 1 protein, a transcriptional regulator and mediator of IFN-gamma-mediated gene expression. The use of cytokines in inducing gp96 levels may have significance in maintaining high levels of gp96 for a sustained immune response.

Anti-tumor effects of PC-SPES, an herbal formulation in prostate cancer.

Prostate cancer is the most common cancer amongst males in developed countries. Surgical removal of the prostate effectively cures the primary disease but the metastatic disease is refractory to most forms of chemotherapy. There is a clinical need to develop novel treatment strategies that exploit the mode of action of both conventional and alternative drugs/medicinal plants. We have been investigating the anti-proliferative and anti-tumor effects of an herbal preparation termed PC-SPES (patent pending, US serial number 08/697, 920) which is a refined powder of eight different medicinal plants. PC-SPES administered as a food supplement caused a dramatic decrease in prostate specific antigen levels in some prostate cancer patients with advanced disease. These preliminary clinical findings laid the foundation for a program to examine the in vitro and in vivo effects of PC-SPES, and identify the active component in this mixture so that a standardized treatment regimen can be formulated. In this communication, we report the anti-tumor effects of PC-SPES incorporated in the diet utilizing a well studied Dunning R3327 rat prostate cancer model. Dietary PC-SPES at levels of 0.05% and 0.025% did not exhibit any toxicity and no significant difference in food intake was noted at the end of six weeks. Dose dependent inhibitory effect of dietary PC-SPES was observed on both tumor incidence (P=0. 01) and rate of tumor growth when tumors were induced in syngeneic Copenhagen rats by intradermal injections of MAT-LyLu cells that are known to metastasize in the lung and lymph nodes. The number of pulmonary metastases in animals on PC-SPES that showed no primary tumor growth had no metastatic lesions in the lung, however, in animals that did not respond to PC-SPES, the number of pulmonary metastases was not significantly different from the non-treated controls. The significant anti-tumor effects of PC-SPES on MAT-LyLu induced tumorigenesis and metastasis in Copenhagen rats, in general refractory to most conventional therapy, suggests a therapeutic benefit of this herbal food supplement and may be a useful adjuvant to conventional therapeutic modalities.

Multiple molecular targets of indole-3-carbinol, a chemopreventive anti-estrogen in breast cancer.

The mechanism of action of the anti-estrogen indole-3-carbinol (I3C), present in cruciferous vegetables, is being examined in our laboratory with a view to promote the use of this naturally occurring chemopreventive as an alternative to synthetic anti-estrogens in human breast cancer. Our previous results clearly demonstrated that despite its low affinity for the estrogen receptor (ER), I3C abrogated estradiol-mediated cellular and biochemical effects in estradiol-responsive cells and tissues. In an earlier report, we identified ER phosphorylation as one of the targets of I3C, and in this communication we describe the consequence of inhibition of ER phosphorylation. Estradiol-induced DNA-binding proteins that bound to several DNA-responsive elements were inhibited by I3C and this effect was not at the level of DNA-protein physical interaction as inclusion of I3C in vitro in the reaction mix did not affect the binding. We analyzed the spectrum of genes induced by estradiol and modulated and/or intercepted by I3C. Our results conclude that although estradiol-mediated functions are affected by I3C, its biochemical targets are multiple and some of these may be modulated by the oligomeric products of I3C.

Proteasome inhibitors differentially affect heat shock protein response in cancer cells.

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.

Preventive and therapeutic effect of tumor derived heat shock protein, gp96, in an experimental prostate cancer model.

Tumor-derived purified heat shock protein (HSP), gp96, has tumor protective effect in a number of experimental cancers that include fibrosarcoma, hepatoma, and spindle cell carcinoma. The rationale for using gp96 as a vaccinating agent stems from the discovery that HSPs, including gp96, chaperone antigenic peptides for eventual recognition and elicitation of an immune response. The immune response generated by the HSP-peptide complex is specific to the tumor from which they are derived. The long-term objective of our studies is to develop a vaccine for primary and metastatic prostate cancer using tumor-derived HSPs. In the present study, we report our results on the tumor protective effect of irradiated Dunning G cells, or purified preparations of g96-peptide complexes as a tumor vaccine. Tumor incidence, latency, and tumor growth were the end points of measurement. Tumor bearing Copenhagen rats, made free of disease by surgical resection of the tumors resisted a fresh challenge of live Dunning G tumor cells. Vaccination with irradiated whole cells failed to elicit any resistance to tumor growth. Vaccination with Dunning G derived purified gp96-peptide complexes delayed both incidence and growth of Dunning G induced tumors. Inhibition of tumor growth was observed when gp96 was administered after tumor induction. Our data suggests that tumor derived gp96-peptide complexes can be used as an effective prophylactic and therapeutic agent even in poorly immunogenic cancer such as prostate cancer. Further investigations will determine specificity of action and define the immunological determinants and experimental conditions for its optimal activity.

Peptide mimotopes of oncoproteins as therapeutic agents in breast cancer.

Generation of an immune response to oncoproteins can lead to a cancer specific protective immunity. Several such oncoproteins are being examined as tumor targets with mixed results. We are evaluating the clinical utility of synthetic peptides that would mimic the antigen immunologically and elicit a tumor specific immune response. HER-2/neu, an oncoprotein whose expression in breast cancer is associated with poor prognosis, lower disease free-survival and a propensity for metastases was chosen as a model. Antibodies, Ab2, Ab4 and Ab5 directed towards the extracellular domain of HER-2/neu were reacted to peptides from two synthetic phage display peptide libraries, LX-8 (12-mer peptide library containing disulfide bridge) and X-15 (linear 15-mer). The isolated peptides were sequenced and characterized for ability to produce high titer antibodies and cross-reactivity. The peptides isolated did not show any sequence homology to protein databases but did show a hierarchy of immunogenic epitopes. Antibodies generated against peptides selected against the same antibody Ab2 or Ab4 showed affinity variation. Phages selected against Ab2 were also able to compete with binding of Ab2 to HER-2/neu. These results validate our hypothesis that synthetic peptides that mimic the antigenic epitope of oncoprotein can be generated and their clinical utility rests on devising a screening mechanism to identify peptides that can elicit an immune response directed to the oncoprotein and if possible its antigenic variants.

Modulation of gene expression by selected fatty acids in human breast cancer cells.

The effect of linoleic acid (LNA, n-6) and eicosopentanoic acid (EPA, n-3) was investigated on the parental MCF-7 cells and those transfected with v-H-ras. Transfection of v-H-ras oncogene renders the normally estrogen dependent MCF-7 cells estrogen independent. The effects of LNA and EPA in both the cell lines were measured by their ability to enhance the steady state cytoplasmic mRNA levels of 1-8 and 2-5 A, two genes whose transcription is enhanced in various cells in response to interferons (IFNs). The modulatory effect of these fatty acids on the level of an oncogene HER-2/neu was also investigated. Our results indicate that both the fatty acids induce mRNA 1-8 in parental MCF-7 cells but not in the ras transfected cell line, although the gene is induced in both cell lines in response to IFNs. mRNA 2-5A was not induced by LNA or EPA in either of the cell lines. HER-2/neu levels were enhanced by EPA in MCF-7-ras cells. Our data provide evidence to support the concept that selected, nutritionally relevant, fatty acids can regulate gene expression in vitro. These fatty acids can also induce second messenger signals similar to the ones generated by IFNs.

HER-2/neu amplification and overexpression in primary human breast cancer is associated with early metastasis.

The etiology of human breast cancer is poorly understood and no specific marker of transformation has been identified. Amplification of HER-2/neu, as reported in a comprehensive study by Slamon et al, was found to be the most powerful predictor of disease-free and overall survival after the status of the axillary lymph nodes. Our study examines the HER-2/neu oncogene in 61 primary human breast cancers at both the DNA level (by Southern blotting) and the protein level (by immunohistochemical methods). Of the 61 tumors analyzed in our study, 17 (28%) had amplification of HER-2/neu. There was no significant correlation of HER-2/neu amplification with age, tumor diameter or hormone receptor status; however, amplification and overexpression of HER-2/neu was significantly correlated with the status of the axillary lymph nodes (P = 0.02). Of 16 patients with amplification of HER-2/neu, 14 (88%) had positive regional nodes. One of the two node negative cases with amplified HER-2/neu had bone marrow micrometastasis. Overall, 16 out of 17 (94%) tumors of the patients having amplified HER-2/neu had metastatic disease at the time of diagnosis. In summary, HER-2/neu amplification is associated with early tumor dissemination in primary human breast cancer and may be a marker of poor prognosis.