A new targeting chemotherapeutic agent, Pt-Mal-LHRH, was synthesized by linking activated cisplatin to luteinizing hormone releasing hormone (LHRH). The compound's efficacy and selectivity toward 4T1 breast cancer cells were evaluated. Carboplatin was selected as the comparative platinum complex, since the Pt-Mal-LHRH malonate linker chelates platinum in a similar manner to carboplatin. Breast cancer and normal cell viability were analyzed by an MTT assay comparing Pt-Mal-LHRH with carboplatin. Cells were also treated with either Pt-Mal-LHRH or carboplatin to evaluate platinum uptake by ICP-MS and cell migration using an in vitro scratch-migration assay. Tumor volume and metastasis were evaluated using an in vivo 4T1 mouse tumor model. Mice were administered Pt-Mal-LHRH (carboplatin molar equivalent dosage) through ip injection and compared to those treated with carboplatin (5 (mg/kg)/week), no treatment, and LHRH plus carboplatin (unbound) controls. An MTT assay showed a reduction in cell viability (p < 0.01) in 4T1 and MDA-MB-231 breast cancer cells treated with Pt-Mal-LHRH compared to carboplatin. Pt-Mal-LHRH was confirmed to be cytotoxic by flow cytometry using a propidium iodide stain. Pt-Mal-LHRH displayed a 20-fold increase in 4T1 cellular uptake compared to carboplatin. There was a decrease (p < 0.0001) in 4T1 cell viability compared to 3T3 normal fibroblast cells. Treatment with Pt-Mal-LHRH also resulted in a significant decrease in cell-migration compared to carboplatin. In vivo testing found a significant reduction in tumor volume (p < 0.05) and metastatic tumor colonization in the lungs with Pt-Mal-LHRH compared to carboplatin. There was a slight decrease in lung weight and no difference in liver weight between treatment groups. Together, our data indicate that Pt-Mal-LHRH is a more potent and selective chemotherapeutic agent than untargeted carboplatin.

ObjectivesCalcium independent group VIA phospholipase A2 (iPLA2β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA2β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.Method and ResultsWe found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA2β dependent manner. These results suggest that iPLA2β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.ConclusionThe present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA2β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.

Currently, 186/188Re and 99mTc are widely used radionuclides for cancer detection and diagnosis. New advancements in modalities and targeting strategies of radiopharmaceuticals will provide an opportunity to enhance imagery and detection of smaller colonies of cancer cells while lowering false-positive diagnoses. To understand the chemistry of agents derived from fac-[99mTc­(CO)3(H2O)3]+ species, the nonradioactive [Re­(CO)3(H2O)3]+ analogue was used. We have designed and synthesized Re-Acdien-LHRH, Re-Acdien-peg-LHRH, and a radiolabeled 99mTc-Acdien-LHRH (rhenium- and technetium-luteinizing hormone-releasing hormone) conjugates using a tridentate linker to detect cancers overexpressing the LHRH receptor. Re-Acdien-LHRH and Re-Acdien-peg-LHRH were synthesized from non-PEGylated and PEGylated LHRH-Acdien, respectively. Cellular uptake of the compounds 99mTc-Acdien-LHRH, Re-Acdien-LHRH, and Re-Acdien-peg-LHRH was found to be significantly enhanced compared to that of untargeted 99mTc alone and unlabeled [Re­(CO)3(H2O)3]+. In addition, the conjugate compounds showed no difference in cellular toxicity compared to untargeted 99mTc alone or unlabeled [Re­(CO)3(H2O)3]+. Further, a competition assay using LHRH indicated selective targeting of Re-Acdien-peg-LHRH toward the LHRH receptor (p < 0.05) compared to that of [Re­(CO)3(H2O)3]+ alone. Together, our data show the design paradigm and synthesis of targeting radionuclides using the LHRH peptide. Our data suggests that utilizing the LHRH peptide can lead to selective targeting and diagnosis of breast cancers expressing the LHRH receptor.

In the United States, breast cancer affects 1 in 8 women during their lifetime. The National Cancer Institute estimated that this means 231,840 new breast cancer cases will arises in 2015, attributing to 40,290 mortalities. Survival of breast cancer and prognosis depends mainly on the type and aggressiveness of the cancer; invasive carcinomas contribute to the majority of cancer related deaths. As such, detection and early stage treatment of breast cancer is crucial to increasing prognosis and favorable outcome. Platinum based chemotherapeutics are widely accepted as a standard treatment for breast cancer. Current drugs on the market, including cisplatin and carboplatin, have been shown to effectively reduce tumor size and improve patient prognosis. However, many patients eventually relapse, developing new tumors that are chemo-resistant, along with exhibiting debilitating side effects, often leading to discontinuation of treatment. To combat this growing problem, we devised and synthesized a new analog of Carboplatin, entitled “Pt-Mal-LHRH”, to selectively target breast cancer cells overexpressing the LHRH receptor. Our initial results utilizing Pt-Mal-LHRH in-vitro showed a higher potency towards 4T1 (stage IV) breast cancer cells compared to those treated with Carboplatin. Cell viability was analyzed by an MTT assay, resulting in significant, (p&gt;0.01), reduction in viable cancer cells treated with Pt-Mal-LHRH compared to those treated with Carboplatin. In addition, drug uptake was evaluated by ICP-MS after cells were treated with either Pt-Mal-LHRH or Carboplatin, displaying a 20-fold increase in 4T1 cellular uptake of Pt-Mal-LHRH compared to Carboplatin. We also found that Pt-Mal-LHRH selectively targets 4T1 breast cancer cells as there was a significant,(p&gt;0.0001), decrease in 4T1 cell viability compared to normal mammary cells. In addition, Pt-Mal-LHRH showed a significant decrease in cell migration using an in-vitro scratch/cell migration assay. Subsequently, using an in-vivo mouse model, our findings showed a significant, (p&gt;0.05), reduction in tumor volume in mice treated with Pt-Mal-LHRH (5 mg/kg/wk) through IP injection compared to those injected with Carboplatin. Furthermore, metastasis and tumor colonization in the lungs was significantly attenuated in Pt-Mal-LHRH treated mice compared to control and Carboplatin treated. Lastly, there was a slight decrease in lung weight and no difference in liver weight between treatment groups. Together, our data indicates that Pt-Mal-LHRH is potentially a more potent and selective chemotherapeutic agent than other platinum based drugs currently on the market, such as Carboplatin. Citation Format: Joseph D. Rollins, Lindsay Calderon, Margaret Ndinguri. Pt-Mal-LHRH, a newly synthesized compound attenuating breast cancer tumor growth and metastasis by targeting overexpression of the LHRH receptor. [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 LB-230.