Journal of Cancer Research
ISSN: 2578-3726 | DOI: 10.64030/2578-3726
Alexandre Tavartkiladze, Russel J. Reiter, Ruite Lou, Dinara Kasradze, Nana Okrostsvaridze, Pati Revazishvili, Maia Maisuradze, Irine Andronikashvili, Pirdara Nozadze, Levan Tavartkiladze, Rusudan Khutsishvili, David Jinchveladze and Givi Tavartkiladze
Background: LUSC shows a strongly glycolytic phenotype with high GLUT1 and LDH-A expression and frequent TP53 mutations [1–6]. Our previous clinical study in triple-negative breast cancer (TNBC) demonstrated that dual targeting of GLUT1 and LDH-A with phloretin and melatonin can reprogram tumor metabolism and induce regression [16]. Here we investigate a more comprehensive three-agent regimen—brassinin, Kevetrin HCl, and melatonin—in LUSC cultures harboring an MTNR1A rs2119882 (−184T/C) promoter polymorphism and mutant TP53, aiming to simultaneously block glycolysis, restore p53 function and normalize MT1 signaling.
Methods: Primary LUSC cultures with rs2119882 (−184T/C) and mutant TP53, and matched normal bronchial squamous epithelium (reference MTNR1A promoter, wild-type TP53) were established. Cells were exposed for 14 days to melatonin + Kevetrin HCl + brassinin (triple), single agents and doublets. Viability, clonogenicity and synergy (Chou–Talalay combination index, CI) were assessed (Table 4, Figure 6) [25]. GLUT1 and LDH-A mRNA/protein were quantified (Figure 3), together with glucose uptake and lactate release. Mitochondrial membrane potential (ΔΨm) was measured by JC-10/JC-1 assays (Figure 2). MTNR1A (MT1) mRNA and protein were evaluated by RT-qPCR and immunofluorescence (IF) (Figure 4). Histone marks (H3K9me3, H3K27ac, H3K4me3) at the MTNR1A promoter were analyzed by ChIP-qPCR. p53 pathway activity was studied by qPCR and Western blotting of p21, PUMA and other targets (Figure 5); TP53 resequencing assessed whether sequence reversion occurred.
Results: The triple regimen produced strong synergy (CI 0.3–0.7 across ED�??�??–ED�??�??) and reduced viability and clonogenicity more than any single agent or doublet (Figure 6, Table 4). GLUT1 and LDH-A expression decreased significantly at both mRNA and protein levels, with concomitant reductions in glucose uptake and lactate release (Figure 3). ΔΨm, which was low at baseline, became restored in triple-treated cells, as indicated by a 2–3-fold increase in JC-10 red/green ratio (Figure 2). p21 and PUMA were robustly up-regulated, cleaved caspase-3 increased, and Annexin V positivity rose, indicating restored p53- dependent apoptosis (Figure 5). In rs2119882-positive LUSC cells, MT1 was weakly detectable at baseline but became strongly expressed after triple treatment, reaching intensities comparable to those in normal epithelium (Figure 4). ChIP-qPCR at the MTNR1A promoter showed a switch from a repressive to an active histone mark profile (H3K9me3↓, H3K4me3/H3K27ac↑). No TP53 sequence reversion was observed, consistent with functional p53 restoration rather than genetic correction.
Conclusions: Combined targeting of STAT3/PI3K/mTOR, p53, MT1 signaling and the GLUT1/LDH-A axis with brassinin, Kevetrin HCl and melatonin produces strong synergistic anti-tumor effects in LUSC, including suppression of glycolysis, mitochondrial repolarization, p53 pathway reactivation and epigenetic normalization of MTNR1A/MT1. In rs2119882-positive cultures this amounts to a partial reverse transformation toward an apoptosis-competent, quasi-normal phenotype. These results justify further in vivo and translational investigation of this triple regimen, with MTNR1A promoter status and p53 functional readouts as candidate biomarkers.