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  1. Short-term hypoxia and nutrient deprivation stress induced shifts in p53 isoform expression in HepG2 tumourspheres
    Keoh LQ, Ahmad Tarmizi MD, Sawai S, Oh L, Ramasamy TS
    Mol Biol Rep, 2026 Jan 10;53(1):271.
    PMID: 41518471 DOI: 10.1007/s11033-025-11415-0
    BACKGROUND: The hypoxic, nutrient-deprived tumour microenvironment (TME), a hallmark of solid tumours, imposes cellular stress that can also paradoxically promote survival and resistance. While TP53 is the most frequently mutated gene in cancer, approximately 60% of hepatocellular carcinoma (HCC) cases retain wild-type TP53 (WTp53), suggesting its isoforms as potential tumorigenic modulators that override canonical tumour-suppressive functions. Therefore, this study aims to delineate p53 isoform profiles in response to short-term hypoxia and nutrient deprivation, recapitulating key stressors in the tumour biology.

    METHOD: We hereby established a 7-day HepG2 tumoursphere model by seeding 15,000 cells/well, which transitioned from normoxia (4 days) to a hypoxic (1% O2), low-serum (1% FBS) (HLS) condition (3 days). Expression of p53 isoforms and downstream targets was assessed.

    RESULTS: The formation of HepG2 tumoursphere at different seeding densities determined the diameter, viability and proliferation profiles, with 15,000 cells/well producing optimal, viable tumourspheres with the highest yield. HLS conditions significantly reduced tumoursphere size, proliferation capacity and viability. Strikingly, multiplex long-amplicon ddPCR revealed substantial upregulation of FLp53α/Δ40p53α, Δ40p53α and Δ133p53α/Δ160p53α mRNA transcripts. While FLp53α/Δ40p53α remained dominant, Δ40p53α and Δ133p53α/Δ160p53α progressively increased, altering the balance among the isoforms. This shift correlated with enhanced expression of the pro-proliferative and survival markers (PCNA and BCL2) and reduced expression of the pro-apoptotic marker (BAX) and cell cycle inhibitor (CDKN1A), suggesting a potential functional role of these isoforms in promoting tumour cell adaptation under stress.

    CONCLUSION: This study highlights stress-induced p53 isoform modulation as a potential survival mechanism in WTp53 HCC in response to TME stress, which warrants further exploration of isoform-specific p53 functions in understanding heterogeneity, resistance and cancer recurrence.

  2. 3D HepG2 spheroid model exhibits drug-specific response profiles validating their application in therapeutic screening
    Choe WL, Ayob AZ, Ahmad Tarmizi MD, Salehen N, Ramasamy TS
    In Vitro Cell Dev Biol Anim, 2026 Sep;62(8):1131-1142.
    PMID: 42479324 DOI: 10.1007/s11626-026-01204-9
    This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC50 values (2.4 µM and 5.3 µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC50 values were determined to be 2.4 µM for doxorubicin and 5.3 µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which ABCB1 was upregulated by doxorubicin, while ABCC2 was selectively induced by sorafenib. Sorafenib progressively reduced BAX/BCL2 ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.
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