Therapeutic dosing and targeting efficacy of Pt-Mal-LHRH towards triple negative breast cancer
Margaret Ndinguri, Lisa Middleton, Jason Unrine, Shu Lui, Joseph Rollins, Emma Nienaber, Cassidy Spease, Aggie Williams, Lindsay Cormier
Abstract
Objective
Pt-Mal-LHRH is a newly synthesized chemotherapeutic agent that was designed to selectively target the luteinizing hormone-releasing hormone (LHRH) receptor expressed by triple negative breast cancer (TNBC). The aim of this study was to evaluate the therapeutic dosing, tumor reduction efficacy, and selective distribution of Pt-Mal-LHRH in-vivo.
Methods and results
LHRH tissue expression levels in-vivo were investigated using western blotting and LHRH was found to be increased in reproductive tissues (mammary, ovary, uterus). Further, Pt-Mal-LHRH was found to have increased TNBC tumor tissue platinum accumulation compared to carboplatin by inductively coupled plasma mass spectrometry analysis.
Introduction
Identification of new chemotherapeutic compounds that target triple negative breast cancer (TNBC) is critical for enhancing patient outcomes and survival rates. About 10–20% of all breast cancers are considered TNBC, in which, cells lack the expression of the estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2/neu) proteins [1]. Due to a lack of hormonal receptor expression, TNBC is rendered unresponsive to many categories of chemotherapy and hormone therapy medications [1]. Subsequently, TNBC is considered the most aggressive and invasive form of breast cancer, leading to high rates of metastasis beyond the breast and the highest rate of recurrence within the first five years after diagnosis [2]. Moreover, women with TNBC have the highest mortality rates within the first five years of diagnosis with a 77% survival rate for TNBC compared to 93% for other types [3]. TNBC is also regarded as the most prevalent form found in young women aged 20–34 years, contributing to 56% of African American and 42% of white women breast cancer cases [4].
Materials and methods
Cell culture
The mouse tumor cell line 4T1 was purchased from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) along with 10% Fetal Bovine Serum, 100 U/mL penicillin and 100 μg/mL streptomycin. They were cultured in a 37ºC tissue culture incubator at 95% humidity and 5% CO2 and passaged until the desired number of cells was reached.
Allograft 4T1 tumor therapeutic dose-range study
Female Balb/c mice, 10wk old were purchased from Jackson Laboratory (Bar Harbor, ME) and housed as previously described by Calderon, et al., 2017 [24]. 4T1 cells (1x106) were suspended in 100uL of DMEM not supplemented with FBS and injected into the right mammary fat pad. After orthotopic tumor initiation (~100mm3) the mice were allocated into treatment groups based on equal tumor volumes and treated with Pt-Mal-LHRH (2.5, 5, 10, or 20 mg/kg) by intravenous injection as day 1 and tumor growth was monitored for 7 days after.
Results
Pt-Mal-LHRH treatment attenuates 4TI TNBC growth
To investigate the safety of therapeutic Pt-Mal-LHRH drug ranges, mice were administered by IV the doses 2.5, 5, 10, or 20 mg/kg. Pt-Mal-LHRH was found to significantly decrease the tumor growth, with 20mg/kg showing the largest amount of tumor regression (Fig 1A). Tumor growth was found to be reduced by 81.7% in response to Pt-Mal-LHRH at the dose of 20 mg/kg (Fig 1A). Further, there was no decrease in mice weight found with Pt-Mal-LHRH dosing indicating drug safety (Fig 1B). In addition, cell blood count analysis showed that Pt-Mal-LHRH administration did not decrease white blood cells, platelets, or red blood cells below normal control, nontumor-bearing mice (Fig 1C–1E). There was a significant increase in white blood cell count in cancer controlled mice compared to the nontumor-bearing mice.
Discussion
The design and synthesis of Pt-Mal-LHRH was developed to enhance the potency and accumulation of platinum within tumors overexpressing the LHRH receptor. Our prior work showed Pt-Mal-LHRH enhanced cytotoxicity at a cellular level due to receptor targeting, along with examining tumor regression through administration of an initial single dose [24]. The current study aimed to expand on our understanding of the Pt-Mal-LHRH compound through investigating the therapeutic dose-range and targeting ability. LHRH is a reproductive hormone that plays a critical role in the hypothalamic-pituitary-gonadal axis and aids in regulating reproduction, puberty, and the release of gonadotropins, through binding to the LHRH receptor in tissues [25]. The expression of the LHRH receptor throughout the body has been previously documented and shown to be expressed at higher levels in reproductive tissues including the mammary, uterus, and ovary [11, 12].
Conclusions
Our results demonstrate that our synthesized Pt-Mal-LHRH chemotherapeutic compound can effectively target and attenuate TNBC tumor growth. This was validated by using two individual TNBC cell line tumor models. Further assessment of Pt-Mal-LHRH treatment showed no decrease in body weight or blood cell count below normal ranges due to toxicity. In addition, the distribution and tissue accumulation results showed targeting of areas overexpressing the LHRH receptor, thus, indicating selectivity towards these tissues while decreasing systemic accumulation, which could potentially mitigate side effects. Taken together, Pt-Mal-LHRH showed enhanced accumulation in reproductive tissues including the ovary, uterus, and mammary compared to carboplatin and effectively targeted TNBC tumors.
Acknowledgments
A special thank you to Charles River Laboratories for conducting and expanding on our experimental studies. Also, we are grateful for the technical inductively coupled plasma mass spectrometry expertise of Shristi Shrestha from the University of Kentucky. All authors contributed equally to the data analysis, conducting experimentation, and overall manuscript conception.
Citation: Ndinguri M, Middleton L, Unrine J, Lui S, Rollins J, Nienaber E, et al. (2023) Therapeutic dosing and targeting efficacy of Pt-Mal-LHRH towards triple negative breast cancer. PLoS ONE 18(10): e0287151. https://doi.org/10.1371/journal.pone.0287151
Editor: Salman Shakil, BRAC University, BANGLADESH
Received: December 31, 2022; Accepted: May 31, 2023; Published: October 10, 2023
Copyright: © 2023 Ndinguri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data is in the Supporting information File.
Funding: Authors: Lindsay Cormier (LC), Margaret Ndinguri (MN); Kentucky Biomedical Research Infrastructure Network (KBRIN) grants (P20GMI04489 and P20GM103436); https://louisville.edu/research/kbrin/kbrin-cores/research-core Yes, Lindsay Cormier: study design, data collection and analysis, decision to publish, and manuscript preparation. Yes, Margaret Ndinguri: study design, data collection and analysis, decision to publish, and manuscript preparation. Authors: Lindsay Cormier (LC), Margaret Ndinguri (MN); Lisa Middleton (LM); KYNETIC grant funding National Institutes of Health U01 HL152392 funded by NCI, NEI, NHGRI, NIDCR, the Commonwealth of Kentucky and administered by NHLBI. https://louisville.edu/research/researchers/innovation-commercialization/translational-grants/kynetic Yes, Lindsay Cormier: study design, data collection and analysis, decision to publish, and manuscript preparation. Yes, Margaret Ndinguri: study design, data collection and analysis, decision to publish, and manuscript preparation. Yes, Lisa Middleton: study design, data analysis, decision to publish, and manuscript preparation.
Competing interests: the authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0287151#abstract0
