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. 2022 Apr 11;14(4):839.
doi: 10.3390/pharmaceutics14040839.

Preclinical Therapeutic Assessment of a New Chemotherapeutics [Dichloro(4,4'-Bis(2,2,3,3-Tetrafluoropropoxy) Methyl)-2,2'-Bipryridine) Platinum] in an Orthotopic Patient-Derived Xenograft Model of Triple-Negative Breast Cancers

Tzu-Chun Kan  1 , Mei-Hsiang Lin  2 , Chun-Chia Cheng  3 , Jeng-Wei Lu  4 , Ming-Thau Sheu  2 , Yuan-Soon Ho  5 , Sri Rahayu  6 , Jungshan Chang  1   7   8
Affiliations

Preclinical Therapeutic Assessment of a New Chemotherapeutics [Dichloro(4,4'-Bis(2,2,3,3-Tetrafluoropropoxy) Methyl)-2,2'-Bipryridine) Platinum] in an Orthotopic Patient-Derived Xenograft Model of Triple-Negative Breast Cancers

Tzu-Chun Kan et al. Pharmaceutics. .

Abstract

Cisplatin is one of the most common therapeutics used in treatments of several types of cancers. To enhance cisplatin lipophilicity and reduce resistance and side effects, a polyfluorinated bipyridine-modified cisplatin analogue, dichloro[4,4'-bis(2,2,3,3-tetrafluoropropoxy)methyl)-2,2'-bipryridine] platinum (TFBPC), was synthesized and therapeutic assessments were performed. TFBPC displayed superior effects in inhibiting the proliferation of several cisplatin-resistant human cancer cell lines, including MDA-MB-231 breast cancers, COLO205 colon cancers and SK-OV-3 ovarian cancers. TFBPC bound to DNA and formed DNA crosslinks that resulted in DNA degradation, triggering the cell death program through the PARP/Bax/Bcl-2 apoptosis and LC3-related autophagy pathway. Moreover, TFBPC significantly inhibited tumor growth in both animal models which include a cell line-derived xenograft model (CDX) of cisplatin-resistant MDA-MB-231, and a patient-derived xenograft (PDX) model of triple-negative breast cancers (TNBCs). Furthermore, the biopsy specimen from TFBPC-treated xenografts revealed decreased expressions of P53, Ki-67 and PD-L1 coupled with higher expression of cleaved caspase 3, suggesting TFBPC treatment was effective and resulted in good prognostic indications. No significant pathological changes were observed in hematological and biochemistry tests in blood and histological examinations from the specimen of major organs. Therefore, TFBPC is a potential candidate for treatments of patients suffering from TNBCs as well as other cisplatin-resistant cancers.

Keywords: PD-L1; apoptosis; autophagy; cell line-derived xenograft model; cisplatin; cisplatin-resistant; patient-derived xenograft; triple-negative breast cancers.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Synthesis of TFBPC.
Figure 1
Figure 1
TFBPC chelated into calf thymus DNA and supercoiled plasmid leading to structural instability. UV-visible absorption spectra of calf thymus DNA with different TFBPC (A) or CDDP (C) concentrations (40–640 μM) after 6 h incubation at 37 °C. The vertical black line demonstrated the maximal absorbance for pure DNA at 260 nm in the absorbance–wavelength plot. The recorded absorbance at 260 nm for a dilution series of TFBPC (B) and CDDP (D) was plotted, and the equation of linear regression line or the second-order polynomial (quadratic) trendline in TFBPC-DNA or CDDP-DNA interaction groups were shown. The gel mobility shift assay for constant plasmid (25 ng/μL) mixed with CDDP or TFBPC for 24 h at 37 °C was applied and three bands (supercoiled, linear, nicked) were identified on 1% agarose gel (E). M and C indicate 1 Kb DNA marker and negative control (plasmid alone) loaded in DNA electrophoresis.
Figure 2
Figure 2
TFBPC inhibited the growth of human cancer cells in a dose-dependent effect. The cytotoxic effects of TFBPC and CDDP on MDA-MB-231 breast cancer cells (A), MCF-7 breast cancer cells (B), COLO205 colon cancer cells (C) and SK-OV-3 ovarian cancer cells (D) were determined using MTT assay after 48 h of incubation. Cell viability was performed in triplicate for at least 5 times independent experiments. Results were represented in percentage and mean ± SD as shown. ** p < 0.01; **** p < 0.0001.
Figure 3
Figure 3
TFBPC and CDDP induced different routes of cell cycle arrest in MDA-MB-231 human breast cancer cells. After MDA-MB-231 cell incubation in 2.5, 5 and 10 μM of CDDP or TFBPC for 24 h, cells were fixed and stained using propidium iodide to detect the DNA content using flow cytometry (A). Proportions of cells at four stages, sub G1, G1, S and G2/M were evaluated using Kaluza Analysis Software and represented as mean ± SD in bar graphs (B,C). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 4
Figure 4
TFBPC activated PARP-mediated apoptosis and autophagy in breast cancers. TFBPC-induced programmed cell death (A) and reactive oxygen species (ROS) expressions (D) were explored using flow cytometry after 15 h and 8 h treatments, respectively. Fractions of cells in the bottom right and top right of quadrants were counted to indicate the proportion of early and late apoptotic cells (B). The percentage of ROS-generating cells (green) gated in the histogram is shown in graph (D). Fragmented DNA extracted from TFBPC- or CDDP-treated MDA-MB-231 cells was examined using DNA electrophoresis test (C) in 1.0% agarose gel. Western blotting of total cell extracts (30 μg of protein) was assessed and quantified for protein expression of apoptosis- and autophagy-related markers (E). The relative expressions of signals in cleaved PARP to full-form PARP (F) and LC3 II to LC3 I (G) were shown. M and C labeled in figures indicate 100 bp DNA ladder marker and DNA extracted from the group without treatment. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; §§§§ p < 0.0001.
Figure 5
Figure 5
TFBPC significantly reduced tumor growth in the TNBC-bearing xenograft model and PDX model. NSG mice (N = 16 in each model) were subcutaneously implanted with MDA-MB-231 cells or patient-derived TNBC tumor tissues on the right flank or mammary gland. In the xenograft model (A–C), mice were intraperitoneally administrated with PBS (5 mL/kg), CDDP (2.11 mg/kg or 5 mg/kg) or TFBPC (5 mg/kg) once per week and sacrificed 28 days after the start of treatments. In the PDX model (D–F), mice were intravenously injected with PBS or platinum-containing agents once per week and sacrificed on day 23. Tumor growth curves and body weight were measured twice per week. Black arrows demonstrated the four times of drug administration. Tumors from xenograft and PDX models were harvested for photography (B,E) and weighed (C,F) to evaluate the compound-dependent anticancer effect. The representative images of PDX models (G) were obtained after sacrificing. Tumors (H) and organs (I) for immunohistochemistry staining of P53, Ki-67, cleaved caspase 3, PD-L1 and hematoxylin and eosin (HE) were harvested from the xenograft model, and all images were carried under 63× magnification. Mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 5
Figure 5
TFBPC significantly reduced tumor growth in the TNBC-bearing xenograft model and PDX model. NSG mice (N = 16 in each model) were subcutaneously implanted with MDA-MB-231 cells or patient-derived TNBC tumor tissues on the right flank or mammary gland. In the xenograft model (A–C), mice were intraperitoneally administrated with PBS (5 mL/kg), CDDP (2.11 mg/kg or 5 mg/kg) or TFBPC (5 mg/kg) once per week and sacrificed 28 days after the start of treatments. In the PDX model (D–F), mice were intravenously injected with PBS or platinum-containing agents once per week and sacrificed on day 23. Tumor growth curves and body weight were measured twice per week. Black arrows demonstrated the four times of drug administration. Tumors from xenograft and PDX models were harvested for photography (B,E) and weighed (C,F) to evaluate the compound-dependent anticancer effect. The representative images of PDX models (G) were obtained after sacrificing. Tumors (H) and organs (I) for immunohistochemistry staining of P53, Ki-67, cleaved caspase 3, PD-L1 and hematoxylin and eosin (HE) were harvested from the xenograft model, and all images were carried under 63× magnification. Mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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