Justin Tang

Acquired chemoresistance, often linked to metabolic adaptation, severely limits therapeutic efficacy in non-small cell lung cancer (NSCLC). Strategies targeting resistance-associated metabolic plasticity are urgently needed. Here, we investigated the combination of Metformin (mitochondrial Complex I inhibitor) and Dichloroacetate (DCA; PDK inhibitor) in chemo-sensitive human A549 NSCLC cells and an acquired Doxorubicin-resistant subline (A549-DR). A549-DR cells displayed significant Doxorubicin resistance (∼15.6-fold; IC50: 3.9 µM vs 0.25 µM in parental A549) and exhibited enhanced glycolysis (higher ECAR) while retaining substantial oxidative phosphorylation (OxPhos) capacity. While single-agent Metformin or DCA showed limited cytotoxicity, their combination induced potent synergistic cell death (Combination Index < 0.5) specifically in A549-DR cells compared to parental A549 (CI ∼ 0.8-1.1). Mechanistically, Metformin inhibited OxPhos, while DCA promoted pyruvate entry into the TCA cycle, preventing compensatory glycolysis. This dual blockade precipitated a catastrophic metabolic collapse uniquely in A549-DR cells, marked by simultaneous suppression of OxPhos (OCR) and glycolysis (ECAR), leading to severe ATP depletion (∼85% reduction), hyperactivation of the energy sensor AMPK, and robust induction of apoptosis (∼58% Annexin V positive). Parental A549 cells were less susceptible, experiencing moderate metabolic inhibition and apoptosis (∼32% Annexin V positive). Our findings highlight a powerful synergistic strategy that exploits the acquired metabolic vulnerabilities of chemoresistant NSCLC cells, inducing an irrecoverable energy crisis and offering a promising therapeutic approach to counteract treatment failure.

Background: Glioblastoma (GBM) remains almost uniformly fatal, owing in part to therapy-resistant cancer stem-like cells (CSCs) and to temozolomide (TMZ) resistance driven by O⁶-methylguanine-DNA methyltransferase (MGMT). Differentiation therapy with all-trans retinoic acid (ATRA) has the potential to attenuate stemness and sensitize GBM to TMZ. We therefore asked whether ATRA reduces expression of key CSC markers and MGMT in established GBM lines. Methods: Two established human GBM cell lines, U87-MG and A172, were cultured under neurosphere-promoting conditions to enrich for potential stem-like subpopulations. Cells were treated with either 1 µM ATRA or vehicle control (DMSO) for 5 days. Total RNA was extracted, and cDNA was synthesized. Quantitative Real-Time PCR (qPCR) assessed relative mRNA expression levels of key stemness transcription factors (SOX2, NES) and the DNA repair gene MGMT and corresponding protein levels were measured by an Enzyme-Linked Immunosorbent Assay (ELISA). Gene expression was normalized to the geometric mean of two validated housekeeping genes (GAPDH, ACTB). Relative quantification was calculated using the ΔΔCt method, and statistical significance was determined using Student’s t-tests. Results: ATRA markedly suppressed stemness and MGMT in both lines. In U87-MG, SOX2 mRNA fell 3.7-fold (p = 0.0008) and protein 2.99-fold (148.3 ± 6.0 → 49.7 ± 2.7 pg µg⁻¹; p = 0.0002); Nestin dropped 4.1-fold (p = 0.0005) and 3.51-fold (450.0 ± 17.3 → 128.3 ± 4.4 pg µg⁻¹; p = 0.00008). MGMT decreased 2.6-fold at transcript level (p = 0.0065) and 2.11-fold at protein level (81.7 ± 4.4 → 38.7 ± 1.8 pg µg⁻¹; p = 0.0005). In A172, SOX2 was reduced 2.9-fold (p = 0.0041) and 2.31-fold (p = 0.0007); Nestin 3.3-fold (p = 0.0028) and 2.79-fold (p = 0.00009). MGMT declined 2.2-fold (p = 0.0132) and 1.82-fold (p = 0.0015), respectively. Conclusions: Five-day exposure to ATRA diminishes SOX2, Nestin, and MGMT at both mRNA and protein levels in stem-enriched GBM cultures, supporting the premise that ATRA-induced differentiation can concurrently blunt CSC traits and TMZ-resistance mechanisms. These data provide a molecular rationale for testing ATRA in combination regimens aimed at improving GBM therapy.

Background: Immune-checkpoint blockade transformed melanoma therapy, yet the B16-F10 model resists PD-1 inhibition. Sub-thermoneutral housing (22°C) causes cold stress, and dosing outside circadian immune peaks dampens anti-tumour immunity. We hypothesised eliminating both would render B16-F10 curable. Methods: C57BL/6 mice (n=40) with B16-F10 tumours were randomised to conventional (22°C; ZT22 PD-1 timing), chronotherapy only (22°C; ZT4), thermoneutrality only (30°C; ZT22), or “double-hit” (30°C; ZT4). Anti-PD-1 was given on days 3, 6, 9, 12. Tumour volume, weight, survival, and day-14 serum IFN-γ were recorded. Synergy threshold Δ ≥ 20% was prespecified. Results: Day 14 control tumours were 1207±48 mm ³ . Chronotherapy and thermoneutrality slowed growth (737±55 mm ³ and 565±28 mm ³ respectively; p3 , –96%; p1 □), yielding seven complete responses and 100% survival. IFN-γ increased 8.8-fold versus control (430±24 vs 48±7 pg mL□ ¹ ; p1 □). Bliss Δ was 25%, confirming synergy. Conclusions: Removing cold stress and circadian mistiming unmasks curative PD-1 response in refractory melanoma. Available warming blankets and morning infusions suggest a practical tactic to rescue PD-1 efficacy in “cold” tumours. A factorial multicentre trial is warranted.

Over the past several decades, viral vector-based vaccines have emerged as some of the most versatile and potent platforms in modern vaccinology. Their capacity to deliver genetic material encoding target antigens directly into host cells enables strong cellular and humoral immune responses, often superior to what traditional inactivated or subunit vaccines can achieve. This has accelerated their application to a wide array of pathogens and disease targets, from well-established threats like HIV and malaria to emerging infections such as Ebola, Zika, and SARS-CoV-2. The COVID-19 pandemic further highlighted the agility of viral vector platforms, with several adenovirus-based vaccines quickly authorized and deployed on a global scale. Despite these advances, significant challenges remain. One major hurdle is pre-existing immunity against commonly used vector backbones, which can blunt vaccine immunogenicity. Rare but serious adverse events, including vector-associated inflammatory responses and conditions like vaccine-induced immune thrombotic thrombocytopenia (VITT), have raised important safety considerations. Additionally, scaling up manufacturing, ensuring consistency in large-scale production, meeting rigorous regulatory standards, and maintaining equitable global access to these vaccines present profound logistical and ethical dilemmas. In response to these challenges, the field is evolving rapidly. Sophisticated engineering strategies, such as integrase-defective lentiviral vectors, insect-specific flaviviruses, chimeric capsids to evade neutralizing antibodies, and plug-and-play self-amplifying RNA approaches, seek to bolster safety, enhance immunogenicity, circumvent pre-existing immunity, and streamline production. Lessons learned from the COVID-19 pandemic and prior outbreaks are guiding the development of platform-based approaches designed for rapid deployment during future public health emergencies. This review provides an exhaustive, in-depth examination of the historical evolution, immunobiological principles, current platforms, manufacturing complexities, regulatory frameworks, known safety issues, and future directions for viral vector-based vaccines.

Background/Objectives: Aberrant expression of c-MYC drives aggressive cancers. A guanine-rich promoter sequence (Pu27) folds into a transcriptionally repressive G-quadruplex (G4). Epigallocatechin gallate (EGCG), the main green tea polyphenol, displays anticancer activity, but clear, easily replicated evidence for direct binding to the c-MYC G4 is lacking. We therefore obtained the first biophysical confirmation of an EGCG–c-MYC G4 interaction using routine UV–visible spectroscopy. Methods: A pre-annealed Pu27 G4 (5 µM) in potassium-rich buffer was titrated with freshly prepared EGCG (0–20 µM) at 25 °C. Full-range UV–Vis spectra (220–400 nm) were recorded after each addition, and absorbance variations at the DNA (260 nm) and ligand (275 nm) maxima were quantified across three independent replicates. Results: EGCG induced pronounced, concentration-dependent hyperchromicity at 260 nm, reaching ~8–10% above baseline at a 4:1 ligand/DNA ratio and exhibiting saturable binding behaviour. Concurrently, the 275 nm band displayed relative hypochromicity coupled with a subtle bathochromic shift. These reciprocal perturbations—absent in buffer-only controls—constitute definitive evidence of a specific EGCG•G4 complex most consistent with external π-stacking or groove engagement rather than intercalation. Conclusions: This study delivers the first rigorous, quantitative UV–Vis confirmation that a readily consumed dietary polyphenol directly targets the c-MYC promoter G4. By marrying conceptual elegance with methodological accessibility, it provides a compelling molecular rationale for EGCG’s anti-oncogenic repertoire, inaugurates an expedient platform for screening G4-reactive nutraceuticals, and paves the way for structural and cellular investigations en route to next-generation c-MYC-directed therapies.

Background: Glioblastoma (GBM) remains a devastating brain malignancy characterized by cellular heterogeneity and therapy resistance. Cancer stem-like cells (CSCs) within GBM are implicated as drivers of tumor progression and resistance to the standard-of-care chemotherapy, temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) expression mediates resistance by repairing TMZ-induced DNA damage. Differentiation therapy offers a potential strategy to reduce the aggressive stem-like phenotype. All-trans retinoic acid (ATRA) is a known differentiating agent. We hypothesized that ATRA treatment could induce differentiation-associated changes in established GBM cell lines, leading to downregulation of stemness markers and the resistance factor MGMT. Methods: Two established human GBM cell lines, U87-MG and A172, were cultured under neurosphere-promoting conditions to enrich for potential stem-like subpopulations. Cells were treated with either 1 µM ATRA or vehicle control (DMSO) for 5 days. Total RNA was extracted, and cDNA was synthesized. Quantitative Real-Time PCR (qPCR) assessed relative mRNA expression levels of key stemness transcription factors (SOX2, NES) and the DNA repair gene MGMT. Gene expression was normalized to the geometric mean of two validated housekeeping genes (GAPDH, ACTB). Relative quantification was calculated using the ΔΔCt method, and statistical significance was determined using Student's t-tests. Results: ATRA treatment (1 µM, 5 days) resulted in a statistically significant downregulation of stemness marker expression in both GBM cell lines compared to vehicle controls. Specifically, SOX2 mRNA levels decreased by 3.7-fold (p=0.0008) in U87-MG and 2.9-fold (p=0.0041) in A172 cells. NES mRNA levels decreased by 4.1-fold (p=0.0005) in U87-MG and 3.3-fold (p=0.0028) in A172 cells. Crucially, ATRA treatment also led to a significant reduction in MGMT mRNA expression. MGMT levels decreased by 2.6-fold (p=0.0065) in U87-MG and 2.2-fold (p=0.0132) in A172 cells following ATRA exposure. Conclusion: Our findings demonstrate that ATRA treatment reduces the expression of stemness-associated genes SOX2 and Nestin in established GBM cell lines cultured under neurosphere conditions. Importantly, ATRA concurrently downregulates MGMT mRNA. These results provide a molecular rationale for exploring ATRA-induced differentiation as a strategy to potentially modulate TMZ resistance pathways in GBM models, warranting further investigation into its therapeutic potential, particularly in combination therapies.

Glioblastoma (GBM) is an exceedingly aggressive primary brain tumor defined by rapid growth, extensive infiltration, and resistance to standard therapies. A central factor driving these malignancies is the subpopulation of glioblastoma stem cells (GSCs), which possess self-renewal capacity, multipotency, and the ability to regenerate tumor heterogeneity. GSCs contribute to key hallmarks of GBM pathobiology, including relentless progression, resistance to chemotherapy and radiotherapy, and inevitable recurrence. GSCs exhibit distinct molecular signatures, enhanced DNA repair, and metabolic adaptations that protect them against conventional treatments. Moreover, they reside within specialized niches—such as perivascular or hypoxic microenvironments—that sustain stemness, promote immunosuppression, and facilitate angiogenesis. Recent discoveries highlight signaling pathways like Notch, Wnt/β-catenin, Hedgehog, STAT3-PARN, and factors such as TFPI2 and HML-2 as critical regulators of GSC maintenance, plasticity, and immune evasion. These findings underscore the complexity of GSC biology and their pivotal role in driving GBM heterogeneity and therapeutic failure. Emerging therapeutic strategies aim to target GSCs through multiple avenues, including surface markers, immunotherapeutics (e.g., CAR T cells), metabolic vulnerabilities, and combination regimens. Advances in patient-derived organoids, single-cell omics, and 3D co-culture models enable more accurate representation of the tumor ecosystem and personalized therapeutic approaches. Ultimately, improved understanding of GSC-specific targets and the tumor microenvironment promises more effective interventions, paving the way toward better clinical outcomes for GBM patients.

The c-MYC oncogene is frequently overexpressed in various human cancers, including aggressive brain tumors like glioblastoma, contributing significantly to tumor cell proliferation and survival. The promoter region of c-MYC contains guanine-rich sequences capable of forming non-canonical DNA structures known as G-quadruplexes (G4s). Stabilization of these G4 structures by small molecule ligands represents a potential therapeutic strategy to downregulate c-MYC expression. Polyphenols, naturally occurring compounds found in plants, possess diverse biological activities, including anticancer effects, and some have been implicated in DNA interactions. Here, we investigate the direct interaction between Epigallocatechin gallate (EGCG), a major polyphenol in green tea, and a synthetic oligonucleotide sequence (Pu27) known to form the predominant G4 structure in the c-MYC promoter. Using simple UV-Vis absorbance spectroscopy, we performed titration experiments by adding increasing concentrations of EGCG to a fixed concentration of pre-formed c-MYC G4 DNA in a potassium-containing buffer. We observed distinct spectral changes upon EGCG addition, notably a concentration-dependent hyperchromicity at the DNA absorbance maximum (~260 nm) and concurrent changes, including hypochromicity and a slight red-shift, near the EGCG absorbance maximum (~275 nm). These spectral perturbations are indicative of a direct interaction between EGCG and the c-MYC G4 DNA structure in vitro. While preliminary, these findings support the hypothesis that EGCG can bind to c-MYC G4 DNA and warrant further investigation into the nature and potential biological significance of this interaction.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, characterized by rapid growth, invasive infiltration into surrounding brain tissue, and resistance to conventional therapies. Despite advancements in surgery, radiotherapy, and chemotherapy, median survival remains approximately 15 months, underscoring the urgent need for innovative treatments. Key considerations informing treatment development include oncogenic genetic and epigenetic alterations that may dually serve as therapeutic targets and facilitate treatment resistance. Various immunotherapeutic strategies have been explored and continue to be refined for their anti-tumor potential. Technical aspects of drug delivery and blood–brain barrier (BBB) penetration have been addressed through novel vehicles and techniques including the incorporation of nanotechnology. Molecular profiling has emerged as an important tool to individualize treatment where applicable, and to identify patient populations with the most drug sensitivity. The goal of this review is to describe the spectrum of potential GBM therapeutic targets, and to provide an overview of key trial outcomes. Altogether, the progress of clinical and preclinical work must be critically evaluated in order to develop therapies for GBM with the strongest therapeutic efficacy.