BACKGROUND:Doxorubicin alone or in combination is widely used as first- or second-line chemotherapy in multiple solid tumors. Yet poor overall/progression-free survival and high mortality reflect the inherent Dox-resistance. Such resistance significantly limits the response to anthracycline-containing regimens. Frequent occurrence of TP53 mutations across cancers suggests that mutant-p53 may drive selective chemoresistance towards first- and second-line chemotherapeutics but the underlying mechanism remains inadequately explored. METHODS:p53 hotspot mutations-R248W, R273H, R175H, R282W and wtp53 were overexpressed in p53-null ovarian (SKOV3) and gastric (KATOIII) cells. R248W mutant was also expressed in p53-null murine breast cancer (4T1) cells. MTT assay, immunoblotting and qPCR were used to assess drug sensitivity, apoptosis, autophagy and gene expression. Flow cytometry and immunofluorescence was used to assess cellular and nuclear doxorubicin levels. RESULTS:p53-mutant expressing cells showed varied responses to cisplatin and doxorubicin. p53-R248W cells were sensitive to cisplatin but highly resistant to doxorubicin as measured by MTT assay and absence of PARP cleavage in multiple cancer cells. All mutants and wtp53 displayed differential degree of doxorubicin efflux. Doxorubicin-resistant p53-R248W SKOV3 cells could retain the drug, possibly due to decreased ABC transporter levels. Reduced BAX and TOP2A and hyperactivated CYP1A1/A2 levels were observed with doxorubicin-treatment in p53-R248W expressing ovarian, gastric and breast cancer cells. CONCLUSION:p53-R248W confers selective doxorubicin resistance to multiple cancer cells through TOP2A suppression and CYP1A1/A2 overexpression. These findings highlight the mutation specific mechanism of doxorubicin resistance that might result in poor response and needs to be considered during treatment plan.

High-Grade Serous Ovarian Cancer (HGSOC) has a dismal five-year survival rate (< 30

Therapy resistance is a complex and multifaceted pathological phenomenon, often driven by mutations in the p53 tumor suppressor gene. This ultimately causes tumor recurrence thereby adversely affecting patient prognosis. Therefore, development of effective chemotherapeutic agents that simultaneously inhibit tumor proliferation and overcome resistance mechanisms is of paramount importance. Recently, we identified Compound 4, a kinetically inert platinum-based antitumor agent capable of bypassing platinum resistance while exhibiting minimal nephrotoxicity. However, its potency in resistance scenario is still not investigated. Herein we evaluate the efficacy of Compound 4 against platinum-resistant cancers driven by mutant p53, using engineered p53-null ovarian (SKOV3) and gastric (KATOIII) cancer cell lines expressing hot-spot p53 mutants (p53mut) as well as patient derived tumor cells. While p53mut expressing cells showed differential sensitivity towards platinum, Compound 4 demonstrated superior cytotoxicity, regardless of p53 mutational status. This enhanced efficacy was attributed to its ability to induce a sustained DNA damage. This was further observed to be compounded by a deficiency in DNA repair responses failing to elicit an effective damage response. Additionally, Compound 4 induced significant mitochondrial depolarization which led to generation of persistent oxidative stress-like environment. Compound 4-mediated oxidative stress plays a crucial role in mediating its robust cytotoxic effects, as pharmacological quenching of reactive oxygen species (ROS) with an antioxidant markedly reduced cell death and gamma H2AX levels. Taken together, these results underscore the potential of Compound 4 as a potent agent capable of overcoming platinum resistance by targeting cancer cells irrespective of their p53 mutational status.

The global incidence of breast cancer has significantly increased, highlighting the need for novel therapeutic strategies. Current treatment options are often limited by drug resistance and adverse effects, necessitating the exploration of alternative compounds. Naringenin, a naturally occurring flavonoid in citrus fruits, exhibits antimicrobial, anti-atherogenic, hepatoprotective, anti-inflammatory, and anticancer properties. This study evaluates the potential of naringenin as an inhibitor of breast cancer cell proliferation. MCF-7 breast cancer cells were used as a model system to assess the anti-proliferative effects of naringenin. Cell viability was evaluated using MTT and colony formation assays, while cell migration was analysed via wound healing assay. Flow cytometry and western blotting were performed to examine cell cycle arrest and apoptosis, and autophagy was assessed through western blotting and confocal microscopy. Naringenin inhibited cellular proliferation in a dose-dependent manner by arresting cells in the S-phase of the cell cycle. It significantly reduced cellular migration and increased early and late apoptosis. Autophagy induction was confirmed by elevated LC3-II expression, p62 degradation, and LC3-II-LAMP1 co-localization. Additionally, C-PARP expression was reduced when cells were co-treated with naringenin and 3-methyladenine (3-MA), indicating pro-apoptotic autophagy. This study demonstrates the anti-migratory and anti-proliferative effects of naringenin and its ability to induce pro-apoptotic autophagy in human breast cancer cells, suggesting its potential as a therapeutic agent.

Resistance to primary chemotherapeutics poses a significant challenge in treating solid tumors. The majority of the second-line chemo and targeted therapeutics act moderately/less effectively in drug-resistant tumors owing to the multicausal nature of drug resistance. Therefore, a single agent with pleiotropic effects would be beneficial in combating this adversity. Withania somnifera exhibits multifunctional anticancer properties, but its role in overcoming chemoresistance remains poorly understood. We evaluated the cytotoxic effect of AshwamaxTM-W. somnifera (WS)-extract and Withaferin A (WFA), in intrinsically resistant (KATO-III and SKOV3) and acquired chemoresistant gastric (AGS5FU) and ovarian (A2780LR) cancer cellular models. We examined their impact on autophagy and apoptosis pathways and elucidated the underlying molecular mechanism. In vivo efficacy of WFA on cisplatin-paclitaxel-resistant epithelial ovarian cancer (EOC) xenografts was assessed using noninvasive optical imaging. Mechanistically, WFA is more proficient in targeting chemoresistant cells than AshwamaxTM-WS extract and activates apoptosis by overriding the AKT-NF-κB-STAT3-survivin axis. Preclinical imaging revealed dose-dependent tumor regression (during and after treatment) in platinum-taxol-resistant EOC xenografts that were unresponsive to cisplatin challenge. WFA, at 3 mg kg-1 dosage, reduced tumor volume by 4.7-fold compared to controls, with sustained antitumor effects persisting after treatment cessation. WFA effectively targets the AKT-NF-κB-STAT3-survivin axis to overcome single and multidrug resistance in gastric and epithelial ovarian cancers, presenting a promising therapeutic alternative for chemoresistant malignancies.

The biggest challenge in developing efficacious anticancer formulations is to improve their targeting specificity and reducing toxicity. For this purpose, HA anchored paclitaxel liposomes (HA-PTX-LIP) were fabricated using synthesized conjugate HA-DSPE (HA Molecular Weight- 12, 76, and 160–200 kDa). Maximum in vitro cell targeting was observed with 160–200 kDa HA-PTX-LIP compared to 11 and 76 kDa HA-PTX-LIP, indicating HA’s MW-dependent HA-CD44 binding and internalization. Further, the effect of grafting density (HA/L ratio- μg HA-DSPE/μmol Lipid) of 160–200 kDa HA-DSPE on tumor targeting was evaluated in vitro at three different levels (low-20 μg/μmol, mid-50 μg/μmol, and high- 100 μg/μmol). HA/L -100 exerted the highest in vitro tumor-targeting potential. Upon in vivo evaluation, 160–200 kDa HA-PTX-LIP (HA/L-100) exhibited 18.11 and 27.4-fold improved AUC and MRT, respectively, compared to the marketed formulation. Moreover, it showed improved biodistribution with less organ AUC (liver, spleen, and kidney) when compared with the marketed formulation. A significant reduction in ROS generation suggested decreased tumor incidence. Compared to the control and marketed formulations, the modified liposome showed increased targeting potential in the in vivo antitumor efficacy on the ovarian tumor model, resulting in a 4.88 and 2.81-fold reduction in tumor volume. Compared to the control and commercial formulations, tumor weight was also reduced by 1.48 and 1.11-fold. Animal survival was 100 percent with 160–200 kDa HA-PTX-LIP (HA/L-100) without significant weight loss. The findings suggest the possibility of rationalizing HA's molecular weight and grafting density for efficient in vivo ovarian tumor targeting.

Many HER2-positive breast cancer (BC) patients relapse within a year of trastuzumab or neratinib treatment. We identified specific pathogenic mutations in the dimerization domains II and IV of the HER2 receptor that contribute to treatment resistance. Mutations G309A, S310Y, and P523S induce significant structural alterations, disrupting crucial HER2:HER2 binding pockets. HER3-preferring mutants exhibited increased HER2:HER3 interactions, as confirmed by proximity ligation assay in HER2-low and HER2-high cell lines. G309A, S310Y, and P523S mutations induced a receptor switch, altering downstream signaling from ERK to AKT activation, leading to high insensitivity to trastuzumab or neratinib in cell survival and migration assays, which was further confirmed by bioluminescence imaging of orthotopic tumors expressing the P523S mutation. This study identifies new hotspot mutations in HER2 domains II and IV causing trastuzumab resistance. Notably, cells with either wild-type or the examined dimerization domain mutations retained sensitivity to the FDA-approved HER2 kinase inhibitor, tucatinib.

Epithelial ovarian cancer (EOC) is a complex disease with diverse histological subtypes, which, based on the aggressiveness and course of disease progression, have recently been broadly grouped into type I (low-grade serous, endometrioid, clear cell, and mucinous) and type II (high-grade serous, high-grade endometrioid, and undifferentiated carcinomas) categories. Despite substantial differences in pathogenesis, genetics, prognosis, and treatment response, clinical diagnosis and management of EOC remain similar across the subtypes. Debulking surgery combined with platinum-taxol-based chemotherapy serves as the initial treatment for High Grade Serous Ovarian Carcinoma (HGSOC), the most prevalent one, and for other subtypes, but most patients exhibit intrinsic or acquired resistance and recur in short duration. Targeted therapies, such as anti-angiogenics (e.g., bevacizumab) and PARP inhibitors (for BRCA-mutated cancers), offer some success, but therapy resistance, through various mechanisms, poses a significant challenge. This comprehensive chapter delves into emerging strategies to address these challenges, highlighting factors like aberrant miRNAs, metabolism, apoptosis evasion, cancer stem cells, and autophagy, which play pivotal roles in mediating resistance and disease relapse in EOC. Beyond standard treatments, the focus of this study extends to alternate targeted agents, including immunotherapies like checkpoint inhibitors, CAR T cells, and vaccines, as well as inhibitors targeting key oncogenic pathways in EOC. Additionally, this chapter covers disease classification, diagnosis, resistance pathways, standard treatments, and clinical data on various emerging approaches, and advocates for a nuanced and personalized approach tailored to individual subtypes and resistance mechanisms, aiming to enhance therapeutic outcomes across the spectrum of EOC subtypes.

BackgroundThe mechanisms enabling dynamic shifts between drug-resistant and drug-sensitive states in cancer cells are still underexplored. This study investigated the role of targeted autophagic protein degradation in regulating ovarian cancer stem cell (CSC) fate decisions and chemo-resistance.MethodsAutophagy levels were compared between CSC-enriched side population (SP) and non-SP cells (NSP) in multiple ovarian cancer cell lines using immunoblotting, immunofluorescence, and transmission electron microscopy. The impact of autophagy modulation on CSC markers and differentiation was assessed by flow cytometry, immunoblotting and qRT-PCR. In silico modeling and co-immunoprecipitation identified ID1 interacting proteins. Pharmacological and genetic approaches along with Annexin-PI assay, ChIP assay, western blotting, qRT-PCR and ICP-MS were used to evaluate effects on cisplatin sensitivity, apoptosis, SLC31A1 expression, promoter binding, and intracellular platinum accumulation in ID1 depleted backdrop. Patient-derived tumor spheroids were analyzed for autophagy and SLC31A1 levels.ResultsOvarian CSCs exhibited increased basal autophagy compared to non-CSCs. Further autophagy stimulation by serum-starvation and chemical modes triggered proteolysis of the stemness regulator ID1, driving the differentiation of chemo-resistant CSCs into chemo-sensitive non-CSCs. In silico modeling predicted TCF12 as a potent ID1 interactor, which was validated by co-immunoprecipitation. ID1 depletion freed TCF12 to transactivate the cisplatin influx transporter SLC31A1, increasing intracellular cisplatin levels and cytotoxicity. Patient-derived tumor spheroids exhibited a functional association between autophagy, ID1, SLC31A1, and platinum sensitivity.ConclusionsThis study reveals a novel autophagy-ID1-TCF12-SLC31A1 axis where targeted autophagic degradation of ID1 enables rapid remodeling of CSCs to reverse chemo-resistance. Modulating this pathway could counter drug resistance in ovarian cancer.

Cancer is a multifaceted disease involving various pathological processes, including uncontrolled proliferation, development of resistance, angiogenesis, metastasis, etc. Therefore, chemotherapeutic agents capable of simultaneously inhibiting proliferation, circumventing chemoresistance, and inhibiting angiogenesis can address multiple aspects of cancer progression. We recently identified a highly promising kinetically inert platinum antitumor agent, namely, Pt-1, that can circumvent cisplatin resistance and showed negligible nephrotoxicity. In this study, we explored the antiangiogenic potential and elucidated the detailed mechanism of cell death through which it exerts its antitumor activity. Pt-1 strongly inhibited angiogenesis in a zebrafish in vivo model at its therapeutically relevant nontoxic dose. Further, Pt-1 exerted antitumor activity through necroptosis- and paraptosis-mediated cell death. Taken together, the combination of antitumor activity with antiangiogenic property in Pt-1 makes it a highly promising antitumor candidate.

The above article, published online on 29 April 2016 in Wiley Online Library (), has been retracted by agreement between the journal Editor-in-Chief, Kevin Ryan, FEBS Press, and John Wiley and Sons Ltd. The retraction has been agreeddue to several image duplications in Figures 4B,E,G, and 5C,D, and inconsistencies between tumour luminescence images and the quantification in Figure 4K. Compelling raw data images were not available from the authors. The editors consider the conclusions substantially compromised and are therefore retracting the paper. Corresponding author Pritha Ray agrees to this retraction, since the raw data are no longer available for analysis and validation. Reference 1 Thakur B, Ray P. p53 loses grip on PIK3CA expression leading to enhanced cell survival during platinum resistance. Mol Oncol. 2016;10(8):1283-1295.

In the original publication [...].

Even in the modern era of precision medicine and immunotherapy, chemotherapy with platinum (Pt) drugs remains among the most commonly prescribed medications against a variety of cancers. Unfortunately, the broad applicability of these blockbuster Pt drugs is severely limited by intrinsic and/or acquired resistance, and high systemic toxicity. Considering the strong interconnection between kinetic lability and undesired shortcomings of clinical Pt drugs, we rationally designed kinetically inert organometallic Pt based anticancer agents with a novel mechanism of action. Using a combination of in vitro and in vivo assays, we demonstrated that the development of a remarkably efficacious but kinetically inert Pt anticancer agent is feasible. Along with exerting promising antitumor efficacy in Pt-sensitive as well as Pt-resistant tumors in vivo, our best candidate has the ability to mitigate the nephrotoxicity issue associated with cisplatin. In addition to demonstrating, for the first time, the power of kinetic inertness in improving the therapeutic benefits of Pt based anticancer therapy, we describe the detailed mechanism of action of our best kinetically inert antitumor agent. This study will certainly pave the way for designing the next generation of anticancer drugs for effective treatment of various cancers.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.

Epithelial ovarian cancer (EOC) is the deadliest gynaecological malignancy and the eighth most prevalent cancer in women, with an abysmal mortality rate of two million worldwide. The existence of multiple overlapping symptoms with other gastrointestinal, genitourinary, and gynaecological maladies often leads to late-stage diagnosis and extensive extra-ovarian metastasis. Due to the absence of any clear early-stage symptoms, current tools only aid in the diagnosis of advanced-stage patients, wherein the 5-year survival plummets further to less than 30%. Therefore, there is a dire need for the identification of novel approaches that not only allow early diagnosis of the disease but also have a greater prognostic value. Toward this, biomarkers provide a gamut of powerful and dynamic tools to allow the identification of a spectrum of different malignancies. Both serum cancer antigen 125 (CA-125) and human epididymis 4 (HE4) are currently being used in clinics not only for EOC but also peritoneal and GI tract cancers. Screening of multiple biomarkers is gradually emerging as a beneficial strategy for early-stage diagnosis, proving instrumental in administration of first-line chemotherapy. These novel biomarkers seem to exhibit an enhanced potential as a diagnostic tool. This review summarizes existing knowledge of the ever-growing field of biomarker identification along with potential future ones, especially for ovarian cancer.

Supplementary Figures S1-S2 from Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects