Carcinoma Research Articles

Composition and Biological Activity of Flavonoid-containing Fractions of an Extract from Gratiola officinalis L.

Gratiola officinalis L. (hedge hyssop), a medicinal plant of the Scrophulariaceae family, has diuretic, purgative, and vermifuge properties. It is used as a herbal tea to treat chronic gastroenteritis, renal colic, jaundice, and intestinal worms. Previously, we have found that an extract from G. officinalis is nontoxic and has antitumor, antioxidant, antimicrobial, antiinflammatory, anticachexic, and other properties. Our aims in this study were to separate the G. officinalis extract into individual fractions, to identify the most biologically active fractions, and to examine the chemical composition of these fractions and their biological activity toward A498 renal carcinoma cells. The G. officinalis extract was fractionated by reversed-phase high-performance liquid chromatography, and each fraction was tested for antitumor activity. The active fractions were characterized by UV-visible electron spectral analysis, circular dichroism analysis, Fourier transform infrared spectroscopy, high-performance liquid chromatography, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy. Two antitumor-active fractions of a flavonoid nature were isolated and chromatographically purified. On the basis of the nuclear magnetic resonance data, the aglycone fragment of the main component of one fraction was found to be structured as 2-(3,4-dimethoxyphenyl)-7-hydroxychroman-4-one, or 3',4'-dimethoxy-7- hydroxyflavanone. The antitumor effect of the most active fraction containing 7-O-glucoside of apigenin, glycoside 7,3'-di-O-luteolin and trace amounts of eupatilin against renal carcinoma A498 cells was manifested in its cytotoxic, cytostatic, apoptotic and autophagosomal activities. In addition, we found 3-(1-2)-glucoside of soyaspogenol B, which is a pentacyclic triterpenoid in the structure.

CIGB-300 internalizes and impairs viability of NSCLC cells lacking actionable targets by inhibiting casein kinase-2 signaling

Overall response rates in advanced Non-Small Cell Lung Cancer (NSCLC) remains low. Thus, novel molecular targets, tailored drugs and/or drug combinations are needed. Casein Kinase-2 (CK2) is a constitutively active and frequently over-expressed enzyme which fosters tumor survival, proliferation and metastasis. By using a clinical-grade and Cell Penetrating Peptide-based inhibitor coined as CIGB-300, we explore the anti-neoplastic effects caused by interruption of CK2 signaling in lung cancer cells lacking EGFR, ALK and ROS mutations. CIGB-300 penetrated and impaired viability and proliferation of Lung Adenocarcinoma (LUAD) (A549, NCI-H522) and Lung Squamous Carcinoma (LUSC) (NCI-H226 and SK-MES-1) cells in a dose-response manner. The differential activity could not be explained by overall peptide uptake or its subcellular distribution, as evidenced by flow cytometry and confocal microscopy. Upon internalization, CIGB-300 interacted with CK2 catalytic subunits (ɑ1/ɑ2) and CK2 substrates, thus impairing phosphorylation of enzyme substrates (CDC37s13, NPM1s125) and downstream proteins (RPS6s325/326). CK2 inhibition induced an early Reactive Oxygen Species (ROS) and mitochondrial membrane depolarization, which predates lung cancer cell death. Finally, intravenous injection of CIGB-300 in a cell line-based xenograft corroborated CIGB-300’s anti-tumor effects and suggested concurrent in situ reductions of CSNK2ɑ subunit and downstream RPS6s235/236 phosphorylation. Overall, CIGB-300 therapeutic hypothesis and antineoplastic effects demonstrated herein, further support the evaluation of this clinical-grade CK2 inhibitor in advanced NSCLC with limited therapeutic options.

Harnessing Nitric Oxide-Donating Benzofuroxans for Targeted Inhibition of Carbonic Anhydrase IX in Cancer.

We describe here the design and antitumor evaluation of benzofuroxan-based nitric oxide (NO)-donor hybrid derivatives targeting human carbonic anhydrases (hCAs) IX and XII. The most effective compounds, 27 and 28, demonstrated potent dual action, exhibiting low nanomolar inhibition constants against hCA IX and significant NO release. Notably, compound 27 showed significant antiproliferative effects against various cancer cell lines, particularly renal carcinoma A-498 cells. In these cells, it significantly reduced the expression of CA IX and iron-regulatory proteins, inducing apoptosis via mitochondrial caspase activity and ferroptosis pathways, as evidenced by increases in ROS, nitrite, and down-regulated expression of ferritin-encoding genes. In three-dimensional tumor models, compound 27 effectively reduced spheroid size and viability. In vivo toxicity studies in mice indicated that the compounds were well-tolerated, with no significant alterations in kidney function. These findings underscore the potential of benzofuroxan-based CA inhibitors for further preclinical evaluations as therapeutic agents targeting renal cell carcinoma.

Phytochemical composition of wild pomegranate juices and their cytotoxicity

PurposeThis study aims to examine the relationship between the chemical composition of juices obtained from fruits of autochthonous wild pomegranate (Punica granatum L.) grown in Montenegro and their cytotoxic effects on cancer cells.Design/methodology/approachTo explore the potential value of wild pomegranate fruits, in vitro biological assays were carried out with juices whose composition was analyzed in detail for sugars, organic acids, vitamin C and phenolic compounds. The effect of juices on survival was determined in human lung A549, cervical HeLa and breast MCF-7 carcinoma cells by MTT assay. As a control, the cytotoxicity against normal fetal lung fibroblasts (MRC-5) was monitored.FindingsAmong cancer cell lines, considering the IC50 related to total phenolics, the lowest value – 13 µg/mL was found for the A549. The strongest effect on lung cells was assumed due to the favorable contribution of ellagitannins to total phenolics in juice as well as the given combination of anthocyanins and their synergistic action. For HeLa cells, the lowest IC50 value was obtained at 88 µg/mL, and the cytotoxicity could be matched with the effects of anthocyanins and catechin. For MCF-7 cells, the lowest IC50 was 504 µg/mL, and the elevated levels of vitamin C and ellagic acid derivatives should have a noticeable effect on these cells.Originality/valueThis study provides an important contribution to the knowledge on the effect of phytochemicals from wild pomegranate juice on lung, cervical and breast cancer cells, in vitro. The present observations suggest that the juice of wild pomegranate has the potential in the fight against cancer.

In silico Screening and in vitro Cytotoxicity Study of Achyranthes aspera Phytochemicals Against Oral Cancer: A Possible Step towards the Development of Anti-cancer Agents.

Oral cancer poses a significant threat to public health worldwide. In addition, because many chemotherapy treatments have negative side effects, natural herbs may be beneficial for oral cancer therapy. Achyranthes aspera (AA), a potential medicinal herb, exerts various pharmacological and biochemical activities. The present study aimed to predict the anti-oral cancer potential of AA using in silico tools and cell death by in vitro testing. A total of fourteen bioactive constituents from AA herb were selected using phytochemical databases. The toxicity of AA herb extract was analysed through MTT assay against oral carcinoma A253 cell line. The binding activities of the phytocomponents against serine/ threonine-specific protein kinases isoforms, namely Akt1 (PDB ID: 3qkk) and Akt2 (PDB ID: 2jdo) proteins, were analysed using Discovery Studio 2021 and PyRx docking software. Cell viability data revealed that AA extract decreased the viability and reduced the number of live cells of the oral carcinoma A253 cell line in a dose-dependent manner. The halfmaximal concentration (IC50) value of AA was assessed as 204.74 μg/ml. Based on binding affinity, saponin C (-CDOCKER energy = -77.9862), oleanolic acid (-CDOCKER energy = - 49.4349), spinasterol (-CDOCKER energy = -38.1246), 36,47-dihydroxyhenpentacontan-4-one (-CDOCKER energy = -32.4386), and 20-hydroxyecdysone (-CDOCKER energy = -31.9138) were identified as the best compounds against Akt1, while, compounds saponin C (-CDOCKER energy = -134.412), oleanolic acid (-CDOCKER energy = -90.0846), spinasterol (-CDOCKER energy = -78.3213), 20-hydroxyecdysone (-CDOCKER energy = -80.1049), and ecdysone (- CDOCKER energy = -73.3885) were identified as Akt2 inhibitors. These top compounds fulfilled drug score values, pharmacokinetic and physicochemical characteristics, and druglikeness parameters. The present findings reveal that the lead phytomolecules of AA could be effective and developed as a prospective drug against oral cancer.

Surgical Outcome and Microbial Colonization of Standardized Smear Locations after Pancreatic Head Resection (Pylorus-Preserving Pancreatoduodenectomy, PPPD) for Chronic Pancreatitis and Pancreatic Head Carcinoma.

Introduction: Patients with chronic pancreatitis (CP) as well as with pancreatic head carcinoma (CA) undergo the surgical intervention named "pylorus-preserving pancreatoduodenectomy according to Traverso-Longmire (PPPD)", which allowed a comparative analysis of the postoperative courses. The hypothesis was that patients with CA would have worse general as well as immune status than patients with CP due to the severity of the tumor disease and that this would be reflected in the more disadvantageous early postoperative outcome after PPPD. Methods: With the aim of eliciting the influence of the different diagnoses, the surgical outcome of all consecutive patients who underwent surgery at the Dept. of General, Abdominal, Vascular and Transplant Surgery at the University Hospital at Magdeburg between 2002 and 2015 (inclusion criterion) was recorded and comparatively evaluated. Early postoperative outcome was characterized by general and specific complication rate indicating morbidity, mortality, and microbial colonization rate, in particular surgical site infection (SSI, according to CDC criteria). In addition, microbiological findings of swabs and cultures from all compartments as well as preoperative and perioperative parameters from patient records were retrospectively documented and used for statistical comparison in this systematic retrospective unicenter observational study (design). Results: In total, 192 cases with CA (68.1%) and 90 cases with CP (31.9%) met the inclusion criteria of this study. Surprisingly, there were similar specific complication rates of 45.3% (CA) vs. 45.6% (CP; p = 0.97) and in-hospital mortality, which differed only slightly at 3.65% (CA) vs. 3.3% (CP; p = 0.591); the overall complication rate tended to be higher for CA at 23.4% vs. 14.4% (CP; p = 0.082). Overall, potentially pathogenic germs were detected in 28.9% of all patients in CP compared to 32.8% in CA (p = 0.509), and the rate of SSI was 29.7% (CA) and 24.4% (CP; p = 0.361). In multivariate analysis, CA was found to be a significant risk factor for the development of SSI (OR: 2.025; p = 0.048); the underlying disease had otherwise no significant effect on early postoperative outcome. Significant risk factors in the multivariate analysis were also male sex for SSI and microbial colonization, and intraoperatively transfused red cell packs for mortality, general and specific complications, and surgical revisions. Conclusions: Based on these results, a partly significant, partly trending negative influence of the underlying disease CA, compared to CP, on the early postoperative outcome was found, especially with regard to SSI after PPPD. This influence is corroborated by the international literature.

Acute exposure to dihydroxyacetone promotes genotoxicity and chromosomal instability in lung, cardiac, and liver cell models.

Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar doses of DHA are cytotoxic, yet the genotoxicity was unclear. We examined the genotoxicity of DHA in cell models relevant to inhalation exposures. Human bronchial epithelial cells BEAS-2B, lung carcinoma cells A549, cardiomyocyte Ac16, and hepatocellular carcinoma HepG3 were exposed to DHA, and low millimolar doses of DHA were cytotoxic. IC90 DHA doses induced cell cycle arrest in all cells except the Ac16. We examined DHA's genotoxicity using strand break markers, DNA adduct detection by Repair Assisted Damage Detection (RADD), metaphase spreads, and a forward mutation assay for mutagenesis. Similar to results for skin, DHA did not induce significant levels of strand breaks. However, RADD revealed DNA adducts were induced 24 h after DHA exposure, with BEAS-2B and Ac16 showing oxidative lesions and A549 and HepG3 showing crosslink-type lesions. Yet, only low levels of reactive oxygen species or advanced glycation end products were detected after DHA exposure. Metaphase spreads revealed significant increases in chromosomal aberrations in the BEAS-2B and HepG3 with corresponding changes in ploidy. Finally, we confirmed the mutagenesis observed using the supF reporter plasmid. DHA increased the mutation frequency, consistent with methylmethane sulfonate, a mutagen and clastogen. These data demonstrate DHA is a clastogen, inducing cell-specific genotoxicity and chromosomal instability. The specific genotoxicity measured in the BEAS-2B in this study suggests that inhalation exposures pose health risks to vapers, requiring further investigation.

Isolation and structure determination of a new bisabolane-type sesquiterpenoid with cytotoxicity from Penicillium oxalicum MZY-202312-521

Endophytic fungi can produce attractive secondary metabolites with various biological activities that have contributed significantly to pharmacotherapy. In this study, three bisabolane-type sesquiterpenoids, including a new one, namely, inonotic acid C (1), together with previously reported compounds (S)-(+)-11-dehydrosydonic acid (2) and sydonic acid (3), were isolated from a marine algal-derived endophytic fungus Penicillium oxalicum MZY-202312-521. Their structures were determined by means of extensive spectroscopic analyses. The absolute configurations of inonotic acid C (1) were established by single-crystal X-ray diffraction method. In vitro cytotoxic experiments on human A549, MCF-7, HeLa, and HepG2 carcinoma cell lines were carried out. The new compound inonotic acid C (1) was found to possess strong inhibitory activity against the MCF-7 cell line, with an IC50 value of 7.7 μM.

Investigating somatic variants and pathways in mismatch repair-deficient (dMMR) colorectal carcinoma in South Africa

AimsColorectal carcinoma (CRC) is a common cause of morbidity and mortality worldwide, and an emerging public health problem in sub-Saharan Africa. Several authors have described an increased frequency of mismatch...

Transformer-based framework for multi-class segmentation of skin cancer from histopathology images.

Non-melanoma skin cancer comprising Basal cell carcinoma (BCC), Squamous cell carcinoma (SCC), and Intraepidermal carcinoma (IEC) has the highest incidence rate among skin cancers. Intelligent decision support systems may address the issue of the limited number of subject experts and help in mitigating the parity of health services between urban centers and remote areas. In this research, we propose a transformer-based model for the segmentation of histopathology images not only into inflammation and cancers such as BCC, SCC, and IEC but also to identify skin tissues and boundaries that are important in decision-making. Accurate segmentation of these tissue types will eventually lead to accurate detection and classification of non-melanoma skin cancer. The segmentation according to tissue types and their visual representation before classification enhances the trust of pathologists and doctors being relatable to how most pathologists approach this problem. The visualization of the confidence of the model in its prediction through uncertainty maps is also what distinguishes this study from most deep learning methods. The evaluation of proposed system is carried out using publicly available dataset. The application of our proposed segmentation system demonstrated good performance with an F1 score of 0.908, mean intersection over union (mIoU) of 0.653, and average accuracy of 83.1%, advocating that the system can be used as a decision support system successfully and has the potential of subsequently maturing into a fully automated system. This study is an attempt to automate the segmentation of the most occurring non-melanoma skin cancer using a transformer-based deep learning technique applied to histopathology skin images. Highly accurate segmentation and visual representation of histopathology images according to tissue types by the proposed system implies that the system can be used for skin-related routine pathology tasks including cancer and other anomaly detection, their classification, and measurement of surgical margins in the case of cancer cases.

Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, and its development comprises a multistep process from intraepithelial neoplasia (IN) to carcinoma (CA). However, the critical regulators and underlying molecular mechanisms remain largely unknown. To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention. A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide (4NQO) to C57BL/6 mice. Moreover, we established a control group without 4NQO treatment of mice. Then, transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses, including low-grade IN (LGIN), high-grade IN (HGIN), and CA, and controlled normal tissue (NOR) samples. Differentially expressed genes (DEGs) were identified in the LGIN, HGIN, and CA groups, and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The CIBERSORT algorithm was used to detect the pattern of immune cell infiltration. Immunohistochemistry (IHC) was also conducted to validate our results. Finally, the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice. Compared with those in the NOR group, a total of 681541, and 840 DEGs were obtained in the LGIN, HGIN, and CA groups, respectively. Using the intersection of the three sets of DEGs, we identified 86 genes as key genes involved in the development of ESCC. Enrichment analysis revealed that these genes were enriched mainly in the keratinization, epidermal cell differentiation, and interleukin (IL)-17 signaling pathways. CIBERSORT analysis revealed that, compared with those in the NOR group, M0 and M1 macrophages in the 4NQO group showed stronger infiltration, which was validated by IHC. Serum cytokine analysis revealed that, compared with those in the NOR group, IL-1β and IL-6 were upregulated, while IL-10 was downregulated in the LGIN, HGIN, and CA groups. Moreover, the expression of the representative key genes, such as S100a8 and Krt6b, was verified in external human samples, and the results of immunohistochemical staining were consistent with the findings in mice. We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions. In addition, we found that macrophage infiltration and abnormal alterations in the levels of inflammation-associated cytokines, such as IL-1β, IL-6, and IL-10, in the peripheral blood may be closely associated with the development of ESCC.

Abstract LB370: Exploring SLC35B family genes in colorectal carcinoma: Bioinformatics and functional insights into prognosis and therapy

Abstract Colorectal carcinoma (CRC) poses a significant health challenge, with recent therapeutic advances yet to substantially improve long-term patient survival. Central to this study is the SLC35B gene family (SLC35B1-4), which encodes nucleotide sugar transporters essential to cellular membranes. Emerging research highlights a link between these transporters' dysfunction and the development and progression of tumors, yet the specific role of the SLC35B family in CRC remains unclear. Our investigation delves into the function of SLC35B genes in CRC, utilizing an integrated bioinformatics approach with databases like GENT2, TCGA, UALCAN, cBioportal, TIMER, and Kaplan-Meier plotter. Our analysis reveals a notable upregulation of SLC35B1/2/4 mRNA in CRC tissues compared to adjacent non-tumor tissues, with a concurrent downregulation of SLC35B3. Promoter methylation analysis shows increased methylation for SLC35B1/3/4, while SLC35B2 demonstrates decreased methylation. According to GEO and TCGA data, CRC patients exhibiting high SLC35B4 expression face lower survival rates. Multivariate Cox regression analysis confirms high SLC35B4 expression as an independent risk factor. Further, SLC35B4 expression correlates with increased infiltration of immune cells, particularly macrophages, and aligns positively with immune checkpoints such as CD274 and HAVCR2. In vitro assays link SLC35B4 to enhanced cancer cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT). In vivo, SLC35B4 knockdown leads to reduced tumor growth in mouse xenograft models. RNA sequencing identifies 241 genes differentially expressed in SLC35B4 knockdown compared to control cells, with Gene Set Enrichment Analysis (GSEA) associating SLC35B4 with the PI3K-Akt signaling pathway. In summary, our research sheds light on the SLC35B gene family's role in CRC, especially SLC35B4, highlighting its potential as both a prognostic marker and a therapeutic target in CRC, particularly through its involvement in the PI3K-Akt signaling pathway. Citation Format: Yu-Jia Chang, Chang-Chun Yang, Cheng-Chin Lee, Chien-Yu Huang. Exploring SLC35B family genes in colorectal carcinoma: Bioinformatics and functional insights into prognosis and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB370.

MicroRNA expression profiling of cutaneous squamous cell carcinomas and precursor lesions

AbstractBackgroundActinic keratoses (AK) are pre‐malignant skin lesions caused by chronic sun exposure. Progression from an AK to intraepidermal carcinoma (IEC) and a cutaneous squamous cell carcinoma (SCC) is well known but the rate of transformation to an invasive SCC is highly variable. Since no definitive biomarkers are available, treatment decisions are made ad hoc.ObjectivesTo fully characterise our AK to SCC progression series, we performed microRNA (miRNA) microarray expression profiling of normal and photodamaged skin, as well as AKs, IEC, and invasive SCCs.MethodsThe study recruited 27 patients who donated fresh biopsies of normal skin, photodamaged skin, AK, IEC, and SCC (n = 67 specimens). All miRbase (v.21) miRNAs were profiled to identify miRNAs related to SCC progression. miRNAs were validated using qRT‐PCR and in vitro phenotypic assays.ResultsThere were 234 robustly expressed miRNAs across the tissue collection, which resulted in 20 miRNA that were differentially expressed ((cor)p ≤ 0.05 and ≥ 10 fold) between normal skin and SCC. Hierarchical clustering all samples illustrated that AKs, IEC, and SCCs were largely indistinguishable, which confirms the premalignant status of an AK. A panel of miRNAs showed significant dysregulation between normal and photodamaged skin and AK. Importantly, we found miR‐34a‐5p and miR‐31‐5p had significant differential expression between AKs and IEC and IEC and SCC respectively. Phenotypic assays determined that the miR‐31 duplex had opposing effects on SCC cell lines which suggests that dysregulation of this duplex may be related to the dynamic control of progression of transformed keratinocytes.ConclusionsThis study confirmed the continuum of AK with IEC and SCC highlighting that miRNA expression plays a role in keratinocyte transformation. Development of our putative miRNA biomarker candidates is warranted to aid in clinical management of patients experiencing high AK load to determine the most appropriate treatment.

Classification of colorectal primer carcinoma from normal colon with mid‐infrared spectra

AbstractIn this project, we used formalin‐fixed paraffin‐embedded (FFPE) tissue samples to measure thousands of spectra per tissue core with Fourier transform mid‐infrared spectroscopy using an FT‐IR imaging system. These cores varied between normal colon (NC) and colorectal primer carcinoma (CRC) tissues. We created a database to manage all the multivariate data obtained from the measurements. Then, we applied classifier algorithms to identify the tissue based on its yielded spectra. For classification, we used the random forest, a support vector machine, XGBoost, and linear discriminant analysis methods, as well as three deep neural networks. We compared two data manipulation techniques using these models and then applied filtering. In the end, we compared model performances via the sum of ranking differences (SRD).

Histology and Lung Nodule Fluorescence in Intraoperative Molecular Imaging With Pafolacianine

BackgroundIntraoperative molecular imaging (IMI) uses a cancer-targeted fluorescent agent injected into patients to localize tumor nodules. Pafolacianine is a folate receptor (FR)–targeted near-infrared fluorescent probe. Almost 10% of patients have false negative fluorescence findings intraoperatively. We hypothesized that tumor histology explains why lung cancer may not fluoresce. MethodsAdenocarcinoma (AC) (A549, LKR) and squamous cell carcinoma (SCC) (H127, H1264) cell lines were stained with pafolacianine. Near-infrared fluorescent microscopy was used to quantify mean fluorescence intensity. Tissue microarray slides of patients with AC and SCC were evaluated by immunohistochemistry for FR alpha (FRα) and beta (FRβ) expression. Finally, we retrospectively analyzed IMI data from clinical trials of patients with AC and SCC receiving pafolacianine. ResultsAC (intensity 30.31) cell lines have a higher fluorescence intensity than SCC cell lines (intensity 5.4) (P < .001). On slide analysis, 93.8% of ACs expressed FRα compared with 44.4% of SCCs (P = .002). Finally, there were 326 patients enrolled in clinical trials: 211 had lesions localized in vivo, and 134 of these patients had pure AC or SCC. All 9 patients with SCC have a positive smoking history and a mean pack-year of 60.2 (SD 3,6), whereas 76% of patients with AC have a history of smoking and a mean pack-year of 29.3 (P = .02). The odds ratio for fluorescence of (AC/SCC) was 2.05 (P = .004) and 2.01 (P = .02) on univariate and multivariate logistic regression, respectively. ConclusionsDuring IMI with pafolacianine, a nonfluorescent nodule is more likely to be SCC than AC. AC has a high probability of fluorescing because of higher expression of FRα or FRβ, or both.

Enhanced extracellular vesicles mediated uttroside B (Utt-B) delivery to Hepatocellular carcinoma cell: Pharmacokinetics based on PBPK modelling

Our prior investigation has confirmed that the anti-hepatocellular carcinoma activity of the plant saponin, specifically Uttroside B (Utt-B), derived from the leaves of Solanum nigrum Linn. This study concentrated on formulating a novel biocompatible nanocarrier utilizing Extracellular vesicles (EVs) to enhance the delivery of plant saponin into cells. The physicochemical attributes of Extracellular Vesicles/UttrosideB (EVs/Utt-B) were comprehensively characterized through techniques such as Transmission Electron Microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Despite the promising therapeutic potential of this uttroside B, mechanistic know-how about its entry into cells is still in its infancy. Our research sheds light on the extracellular vesicle-mediated mechanism facilitating the entry of the saponin into cells, a phenomenon confirmed through the use of by confocal microscopy. We further analysed drug-releasing kinetics and simulated the Pharmacokinetics by PBPK modelling. The simulated pharmacokinetics revealed the bioavailability of Uttroside-B in oral administration against intravenous administration.

Multifaceted approach toward mapping out the anticancer properties of small molecules via invitro evaluation on melanoma and nonmelanoma skin cancer cells, and in silico target fishing.

Melanoma and nonmelanoma skin cancers are among the most prevalent and most lethal forms of skin cancers. To identify new lead compounds with potential anticancer properties for further optimization, invitro assays combined with in-silico target fishing and docking have been used to identify and further map out the antiproliferative and potential mode of action of molecules from a small library of compounds previously prepared in our laboratory. From screening these compounds invitro against A375, SK-MEL-28, A431, and SCC-12 skin cancer cell lines, 35 displayed antiproliferative activities at the micromolar level, with the majority being primarily potent against the A431 and SCC-12 squamous carcinoma cell lines. The most active compounds 11 (A431: IC50 = 5.0 μM, SCC-12: IC50 = 2.9 μM, SKMEL-28: IC50 = 4.9 μM, A375: IC50 = 6.7 μM) and 13 (A431: IC50 = 5.0 μM, SCC-12: IC50 = 3.3 μM, SKMEL-28: IC50 = 13.8 μM, A375: IC50 = 17.1 μM), significantly and dose-dependently induced apoptosis of SCC-12 and SK-MEL-28 cells, as evidenced by the suppression of Bcl-2 and upregulation of Bax, cleaved caspase-3, caspase-9, and PARP protein expression levels. Both agents significantly reduced scratch wound healing, colony formation, and expression levels of deregulated cancer molecular targets including RSK/Akt/ERK1/2 and S6K1. In silico target prediction and docking studies using the SwissTargetPrediction web-based tool suggested that CDK8, CLK4, nuclear receptor ROR, tyrosine protein-kinase Fyn/LCK, ROCK1/2, and PARP, all of which are dysregulated in skin cancers, might be prospective targets for the two most active compounds. Further validation of these targets by western blot analyses, revealed that ROCK/Fyn and its associated Hedgehog (Hh) pathways were downregulated or modulated by the two lead compounds. In aggregate, these results provide a strong framework for further validation of the observed activities and the development of a more comprehensive structure-activity relationship through the preparation and biological evaluation of analogs.

Abstract B143: HRO761, a first-in-class, clinical stage WRN inhibitor with potent preclinical anti-tumor activity in MSIhigh models

Abstract Colorectal carcinoma (CRC) as well as other indications that have lost competence in mismatch repair and have a phenotype known as microsatellite instability (MSI), remain a significant unmet medical need. Large-scale functional genomics screens across cell line panels have identified the Werner Syndrome RecQ helicase (WRN) as being synthetic lethal with MSIhigh cancers. Thus, WRN inhibitors may offer a new therapeutic avenue in MSIhigh cancers. We report the preclinical characteristics of HRO761, a first-in-class, highly potent and selective inhibitor of WRN helicase, with optimized drug-like properties. We evaluated the pharmacokinetics and pharmacodynamics of HRO761 when administered orally once daily in MSIhigh and also assessed the efficacy as single agent across cell (CDX) and patient-derived (PDX) xenografts as well as in combination with irinotecan, a prodrug derivative of camptothecin which acts as topoisomerase I inhibitor. Daily oral administration of HRO761 was highly efficacious across the CDX and PDX models evaluated with a disease control rate of 70% across all indications. In the SW48 CDX model, tumor regression was sustained for 2 months at the highest dose followed by slow relapse. HRO761 also induced WRN degradation, activated the DNA damage response and induced p53 target genes in a dose dependent manner. In vitro combination with irinotecan deepened sensitivity to HRO761 with a more sustained response in MSIhigh CRC cell lines. Combining administration of HRO761 daily with irinotecan weekly was highly efficacious and beneficial over single agent in CDX and PDX models. In CDX models, the combination showed a robust benefit, translating into homogeneous, sustained regression leading to a dose-dependent tumor growth delay even after treatment discontinuation. Overall, we demonstrate that the novel, selective, first-in-class WRN inhibitor HRO761 is a promising therapeutic approach for the treatment of MSIhigh cancer patients either as single agent or in combination with irinotecan. Clinical development is currently ongoing to assess the safety, tolerability and preliminary anti-tumor activity in patients with MSIhigh colorectal cancer and other MSIhigh solid tumors [clinicaltrials.gov NCT05838768]. Citation Format: Stephane Ferretti, Isabel Jaco, Andrea Decker, Christelle Hemmerlin, Clemens Scheufler, Cornelia Quadt, Dario Sterker, Elena Gavioli, Eloisa Jimenez Nunez, Ernesta Dammassa, Fanny Schaeffer, Genevieve Albrecht, Giorgia Clementi, Hansjoerg Martus, Jacques Hamon, Juergen Hinrichs, Laurent Laborde, Marion Dourdoigne, Michele Moschetta, Ramona Stump, Rita Andraos-Rey, Sarah Welly, Stephanie Barbe, Vincent Romanet, Markus Reschke, Ruben De Kanter, Michael Jensen, Henrik Moebitz, Marta Cortes Cros. HRO761, a first-in-class, clinical stage WRN inhibitor with potent preclinical anti-tumor activity in MSIhigh models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B143.

Abstract A040: Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR

Abstract The epidermal growth factor receptor (EGFR), an established oncology target, is a transmembrane protein highly expressed on the surface of malignant tumors such as glioblastoma, lung, liver, bladder and breast cancers. In addition to overexpression, EGFR mutants are frequently associated with malignancy. For example, EGFR variant III (EGFRvIII), deletion of exons 2-7, is only expressed on cancer cells and is associated with poor prognosis and chemotherapy resistance. To target cancer-expressed EGFR, including EGFRvIII, we generated the 40H3 monoclonal antibody which is reactive with the 287-302 loop that is fully exposed on the extracellular domain of EGFRvIII. The 40H3 antibody also showed strong reactivity for cells expressing gene-amplified EGFR but little or no reactivity for cells expressing normal levels of wild type EGFR. When conjugated with toxic payloads (either tesirine or deruxtecan), the resulting antibody-drug conjugates (ADCs) exhibited antitumor activity in several xenograft models. To improve its utility as a clinical candidate, we humanized the variable portions of the heavy and light chains of 40H3, without changing the original CDRs. The variable regions of 40H3, heavy and light chains, were compared to the closest human immunoglobulin families and then framework residues were altered to produce candidate humanized antibodies. The humanized variable chains were then fused with a human IgG1 heavy chain and a kappa light chain to generate full-sized humanized antibodies. A total of 9 humanized antibodies were generated by pairing 3 variable humanized heavy chains (VH1, VH2, VH3) with 3 variable humanized light chains (VL1, VL2, VL3). To evaluate the binding activity of humanized antibodies, we used either immobilized EGFRvIII (by ELISA or Octet) or various cancer cells expressing either EGFR or EGFRvIII (by flow cytometry). All humanized antibodies retained binding activity to immobilized EGFRvIII but bound with different apparent affinities to cells overexpressing EGFR or cells transfected with EGFRvIII. The ‘A10’ antibody was the best candidate. Mutations introduced in A10 preserved the binding affinity of the 40H3 antibody but exhibited an increased affinity compared to the corresponding chimeric antibody constructed with the original mouse VH and VL. The other eight antibodies retained EGFRvIII binding activity, albeit with a lower cell binding affinity. Specifically, A10 showed improved binding to cells with EGFR gene amplification including two TNBC lines, MDA-MB-468 and BT-20, epithelial cancer cells, A431, head and neck squamous carcinoma, UMSCC17B, as well to cells expressing EGFRvIII, F98npEGFRvIII. No binding of A10 was detected to cell lines expressing wild type EGFR, WI-38 or U87MG. In summary, humanized candidates of the 40H3 monoclonal antibody retained EGFR binding. Of 9 antibodies, A10 exhibited the best cell binding activity elevating it to our top clinical candidate for the production of ADCs or similar antibody-based therapeutics. Citation Format: Tamara G Fernandes Costa, Robert Sarnovsky, Maria Teresa Bilotta, Antonella Antignani, David FitzGerald. Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A040.

Activation of G-Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces Progression of Vulvar Carcinoma Cells.

Whether G protein-coupled estrogen receptor 1 (GPER1) is tumor-promoting or tumor-suppressive depends in part on tumor entity. Little is known about the function of GPER1 in vulvar carcinoma. In this work, we aim to clarify what role GPER1 plays in vulvar cancer, tumor-promoting or tumor-suppressive. Localization of GPER1 in A431 and CAL-39 vulvar carcinoma cells was examined by immunofluorescence. Using a tissue microarray of vulvar neoplasias, the correlation between GPER1 expression and grade of malignancy was investigated. A431 and CAL-39 cells were treated either with GPER1 agonist G1 or antagonist G36. Proliferation was quantified by BrdU assay and viability examined using Resazurin assay. Morphological changes were analyzed by microscopy and measured using ImageJ. Cell migration was analyzed by gap closure assay. Clonogenic potential was tested by colony and sphere formation. Expression of estrogen receptors was examined by Western blot. GPER1 was found consistently expressed in vulvar neoplasia tissues. The immune-reactive score was found to be significantly higher in tissue samples of lymph node metastases and neoplasias with grade 3. In A431 and CAL-39 vulvar carcinoma cells, GPER1 expression was mainly found in the cytoplasm and nuclei. Treatment of A431 and CAL-39 cells with GPER1 agonist G1 resulted in a decrease in proliferation and migration. In addition, colony formation and tumor sphere formation were reduced. Furthermore, morphological signs of necrosis and reduction in cell viability after G1 treatment were observed. The GPER1 antagonist G36 did not have significant effects on vulvar carcinoma cells. Neither agonist G1 nor antagonist G36 treatment resulted in altered expression of estrogen receptors. Activation of GPER1 with GPER1 agonist G1 reduces the tumorigenic potential of the vulvar carcinoma cells. It can be deduced from this that GPER1 appears to have a tumor-suppressive effect in vulvar carcinoma.