Evaluation Of The Effectiveness Of Adjuvant Immunotherapy Combined Use With Epigenetic Agent In Conventional Treatment Of Triple Negative Breast Cancer
Project no. 24010 · Status: Kapanmış (Yayın bekleniyor) · 2023
Summary
<p>Triple-negative breast cancer (T<nBC) is a treatment-resistant type of breast cancer known for its high metastasis rate and poor prognosis. The combination of the PD-L1 inhibitor Atezolizumab and Nab-paclitaxel, which consists of albumin-bound paclitaxel nanoparticles, is used in current treatment protocols but does not provide the expected benefit in most patients. Epigenetic agents are considered as potential cancer treatment agents by inducing apoptosis and cell cycle arrest in cancer cells and by their immunomodulatory effects. Since HDAC 1, 6 and 8 genes play a role in breast cancer progression, Droxinostat, an HDAC 3, 6 and 8 inhibitor, may be an effective approach to overcome chemotherapeutic and immunotherapeutic resistance. In this study, the effects of Droxinostat were compared with Nab-paclitaxel and Atezolizumab. The cytotoxic effects of single doses as well as double and triple combinations of Droxinostat, Atezolizumab and Nab-paclitaxel on MDA-MB-231 TNBC and MCF-10A healthy breast cells were evaluated by XTT method and IC50 and ED50 doses were calculated. Apoptotic effects were analyzed by Annexin V method and apoptosis and cell cycle analysis were performed by flow cytometry. In addition, wound healing assay and invasion analyses were performed, expression of Ki-67 protein was determined by immunofluorescence method, oxidative stress markers were measured and transcriptional expression of 26 genes involved in the carcinogenesis process were analyzed by q-RT-PCR. The results show that the IC50 dose of Droxinostat was determined as 60 µM in MDA-MB 231 cells and 190 µM in MCF-10A cells at 72 hours. The IC50 dose of Nab-paklitaksel was 0.58 µM in MDA-MB-231 cells and 0.16 µM in MCF-10A cells, and the combination doses of 48 µM for Droxinostat and 0.28 µM for Nab-paklitaksel showed a moderate synergistic </p><p>effect with a CI index of 0.808. Since Atezolizumab did not show toxic effects, 100 nM dose was used in all experiments. It was observed that mono and combination doses of Droxinostat increased apoptosis and necroptosis 5-6 fold in MDA-MB-231 cells, whereas it only increased necroptosis in MCF-10A cells. Droxinostat caused cell death by activating both apoptosis and necroptosis pathways. Moreover, Droxinostat was found to arrest the cell cycle more in the S and M phases, whereas Nab-paclitaxel induced more cell retention in the M phase. The agents did not cause significant changes in healthy cells. 5 A decrease in Ki-67 expression was observed, especially in MDA-MB-231 cells. The agents decreased both the number of invasive cells and cell migration. Droxinostat had minimal effect on antioxidant biomarkers but induced ROS in combination with Nab-paclitaxel. Gene expression analysis revealed that Atezolizumab decreased PD-L1 expression by 6.33-fold in </p><p>MDA-MB-231 cells, while Droxinostat increased this expression and showed no change in MCF-10A cells. Furthermore, Atezolizumab and Nab-paclitaxel were found to increase PD1 expression approximately 2-fold in MDA-MB-231 cells. Droxinostat was found to inhibit HDAC 8 the most. Both single and combination doses of droxinostat increased the expression levels of caspase-3 and caspase-8. Nab-paclitaxel also increased the expression of these genes, but this increase was more limited compared to Droxinostat. The fact that the IC50 dose of Droxinostat in healthy cells was 3 times higher than in cancer cells, that it increased apoptosis and necroptosis more than Nab-paclitaxel and Atezolizumab, and its potential as an effective molecule indicate that it can be used in the treatment of TNBC.</p>