Proteomic Analysis of Butyrate-Resistant Colorectal Cancer-Derived Exosomes Reveals Potential Resistance to Anti-Cancer Drugs.

BACKGROUND: Butyrate-resistant (BR) cells play an important role in acquiring chemoresistance in colorectal cancer (CRC). Our previous study demonstrated that BR CRC cells showed cross-resistance to chemotherapy drugs, including 5-fluorouracil and oxaliplatin, in both monolayer and spheroid cultures. The mechanisms underlying drug resistance were also elucidated. However, the link between parental (PT) and BR cells remains unclear. Extracellular vesicles (EVs) are key cell-cell communications that transport various molecules, including DNA, RNA, and proteins, between the donor and target cells. EVs contribute to drug resistance in cancers, such as melanoma and lung cancer. Recently, we focused on the correlation of proteomic profiles of EVs from different cell types. METHODS: In this study, we analyzed the proteomic profiles of EVs derived from PT and BR cells to investigate the mechanisms underlying the butyrate- and chemo-resistant phenotypes. EVs were isolated from PT and BR cells using ultracentrifugation. The characteristics of the EVs were evaluated using western blot and transmission electron microscopy. The EV proteomic data were further analyzed using liquid chromatography-mass spectrometry. RESULTS: We identified a unique protein expressed in BR cells related to the chemoresistant phenotype. Functional enrichment analysis showed that BR cells had higher protein catalytic activity, binding, and transcription activity. The STITCH database showed a greater correlation between protein-drug interactions in BR cells than in PT cells. Moreover, our findings support the hypothesis that EVs promote tumor progression and metastasis and affect the tumor microenvironment. CONCLUSIONS: Proteomic analysis of EVs from BR CRC cells reveals insights into drug resistance mechanisms, including protein-mediated carcinogenesis and reduced drug uptake, offering potential strategies to overcome resistance in clinical practice.

Proteomic analysis of small extracellular vesicles unique to cervical cancer.

Background: Cervical cancer (CC) is the fourth most common cancer in females worldwide. Existing biomarkers for CC, such as squamous cell carcinoma antigens, show low specificity. Hence, a novel biomarker for the diagnosis of CC is required. Through proteomic analysis, this study aimed to distinguish between the small extracellular vesicle (sEV) protein profiles of healthy controls (HC) and CC sera and to identify potential sEV proteins that can serve as biomarkers for CC diagnosis. Methods: The number and size distribution of sEVs in HC and CC sera were measured using nanoparticle tracking analysis. Differential ultracentrifugation combined with size-exclusion chromatography was used to isolate and purify sEVs. Liquid chromatography-tandem mass spectrometry was used to identify and compare the protein profiles between patients with CC and HC. Differentially expressed extracellular vesicle (EV) proteins were validated using The Cancer Genome Atlas database. Results: The EV particle concentration in patients with CC was marginally higher than that in HC. Proteomic and functional protein analyses revealed a difference in the EV protein profiles between HC and CC and identified proteins that can serve as biomarkers for CC. Conclusions: This study provides insights into the potential of sEVs as less invasive biomarkers for CC diagnosis. Validation with a well-designed cohort should be performed to determine the clinical diagnostic value of specific protein markers for CC.

Pretreatment total lymphocyte count as a prognostic factor of survival in patients with recurrent cervical cancer after definitive radiation-based therapy: a retrospective study.

OBJECTIVE: This study evaluated the association between pretreatment total lymphocyte count (TLC) and overall survival (OS) in patients with recurrent cervical cancer. METHODS: We retrospectively reviewed 290 patients with recurrent cervical cancer with definite complete responses to either definitive radiotherapy or concurrent chemoradiotherapy between January 2009 and December 2022. The associations between pretreatment TLC and progression-free survival (PFS) and OS rates were evaluated. RESULTS: Ninety-three patients (32%) had a pretreatment TLC <1,000 cells/mm3. Patients with a pretreatment TLC <1,000 cells/mm3 had lower treatment response rates than their counterparts (P=0.045). The OS and PFS rates were significantly higher in patients with pretreatment TLC ≥1,000 cells/mm3 than in those with pretreatment TLC <1,000 cells/mm3 (10.74 vs. 3.89 months, P<0.0001; 8.32 vs. 4.97 months, P=0.042; respectively). Moreover, pretreatment TLC ≥1,000 cells/mm3 was identified as an independent prognostic factor for OS in both univariate analysis (hazard ratio [HR], 0.57; 95% conficence interval [CI], 0.44-0.74; P<0.001) and multivariate analysis (HR, 0.64; 95% CI, 0.47-0.86; P=0.003). However, TLC ≥1,000 cells/mm3 was identified as a prognostic factor for PFS only in univariate analysis (HR, 0.71; 95% CI, 0.51-0.99; P=0.043) but not in the multivariate analysis (HR, 0.81; 95% CI, 0.55-1.18; P=0.3). CONCLUSION: Pretreatment TLC was associated with treatment response and was identified as an independent prognostic factor associated with the survival outcomes of patients with recurrent cervical cancer.

Association between Resting Heart Rate and Oncological Outcomes in Patients with Early-Stage Cervical Cancer Who Have Undergone Radical Hysterectomy with Pelvic Lymphadenectomy.

INTRODUCTION: A few studies have explored the association of resting heart rate (RHR) with mortality and/or other oncological outcomes in patients with specific cancers such as breast, colorectal, and lung cancer. This study aimed to evaluate the association between the RHR and oncological outcomes in patients with early-stage cervical cancer (CC) who underwent radical surgical resection. METHODS: We included 622 patients with early-stage CC (stages IA2-IB1). The patients were divided into four groups based on the RHR as follows: quartile 1, ≤64; quartile 2, 65-70; quartile 3, 71-76; and quartile 4, >76 beats per min (bpm), with the lowest quartile being the reference group. We evaluated the associations of the RHR and clinicopathological features with oncological outcomes using Cox proportional-hazards regression. RESULTS: There were clear among-group differences. Further, there was a significant positive correlation of RHR with tumor size and deep stromal invasion. Multivariate analysis revealed that RHR was an independent prognostic factor for disease-free survival (DFS) and overall survival (OS). Compared with patients with an RHR ≤70 bpm, those with an RHR of 71-76 bpm had a 1.84- and 3.05-times higher likelihood of DFS (p = 0.016) and OS (p = 0.030), respectively, while those with RHR >76 bpm had a 2.20-times higher likelihood of DFS (p = 0.016). CONCLUSION: This is the first study to demonstrate that RHR may be an independent prognostic factor for oncological outcomes in patients with CC.

Characterization of Butyrate-Resistant Colorectal Cancer Cell Lines and the Cytotoxicity of Anticancer Drugs against These Cells.

Colorectal cancer (CRC) is the third most common cancer worldwide. The gut microbiota plays a critical role in homeostasis and carcinogenesis. Butyrate, a short-chain fatty acid produced by the gut microbiota, plays a role in intestinal homeostasis and acts as an anticancer agent by inhibiting growth and inducing apoptosis. However, microbiota studies have revealed an abnormally high abundance of butyrate-producing bacteria in patients with CRC and indicated that it leads to chemoresistance. We characterized butyrate resistance in HCT-116 and PMF-K014 CRC cells after treatment with a maximum butyrate concentration of 3.2 mM. The 50% inhibitory concentration of butyrate was increased in butyrate-resistant (BR) cells compared with that in parental (PT) cells. The mechanism of butyrate resistance was initially investigated by determining the expression of butyrate influx- and drug efflux-related genes. We found the increased expression of influx- and efflux-related genes in BR cells compared with that in PT cells. Proteomic data showed both identical and different proteins in PT and BR cells. Further analysis revealed the crossresistance of HCT-116 cells to metformin and oxaliplatin and that of PMF-K014 cells to 5-fluorouracil. Our findings suggest that the acquisition of butyrate resistance induces the development of chemoresistance in CRC cells, which may play an important role in CRC development, treatment, and metastasis.

Cytotoxic effect of metformin on butyrate-resistant PMF-K014 colorectal cancer spheroid cells.

Three-dimensional (3D) cell culture models are used in cancer research because they mimic physiological responses in vivo compared with two-dimensional (2D) culture systems. Recently, cross-resistance of butyrate-resistant (BR) cells and chemoresistance in colorectal cancer (CRC) cells have been reported; however, effective treatments for BR cells have not been identified. In this study, we investigated the cytotoxicity of metformin (MET), an anti-diabetic drug, on BR CRC cells in a 3D spheroid culture model. The results demonstrate that MET decreases spheroid size, migration, and spheroid viability, while it increases spheroid death. The molecular mechanism revealed that AMP-activated protein kinase (AMPK) and Akt serine/threonine kinase 1(Akt) were significantly upregulated, whereas the acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated, which led to caspase activation and apoptosis. Our findings show the potential cytotoxicity of MET on CRC-BR cells. The combination of MET and conventional chemotherapeutic drugs should be addressed in further studies to reduce the side effects of standard chemotherapy for CRC.

Predictive Value of the Hemoglobin-Albumin-Lymphocyte-Platelet (HALP) Index on the Oncological Outcomes of Locally Advanced Cervical Cancer Patients.

Purpose: The oncological outcomes of locally advanced cervical cancer (LACC) patients after treatment are poor and heterogeneous. This study aimed to determine the role of the hemoglobin-albumin-lymphocyte-platelet (HALP) inflammatory index in predicting oncological outcomes in LACC patients. Patients and Methods: A total of 1588 LACC patients who received radiation therapy or concurrent chemoradiation were divided into training and test sets. Characteristics, survival, and a HALP cutoff determined by X-tile software were used to build predictive survival models on the training data. Validation of the model was performed on both sets. Results: Patients with a HALP score ≤22.2 tended to have lower age (p < 0.001), lower comorbidity rate (p = 0.016), lower body mass index (p < 0.001), higher stage (p < 0.001), larger tumor size (p < 0.001), and higher likelihood to receive radiation alone than concurrent chemoradiation (p < 0.001). Survival analysis demonstrated that HALP >22.2 was independently associated with better progression-free survival (PFS; hazard ratio; HR 0.55) and overall survival (OS; HR 0.43). Validation of survival prediction by receiver-operating characteristics demonstrated a significantly improved area under the curve of survival prediction in both sets (p < 0.001) after the addition of the HALP index to the model. Conclusion: A lower HALP score was an independent predictive factor for poorer oncological outcomes. The addition of the HALP index can improve the accuracy of predicting the oncological outcomes of LACC patients.

Molecular insights and clinical impacts of extracellular vesicles in cancer.

Cell-to-cell communication is a pivotal aspect of cancer biology. Recently, extracellular vesicles (EVs)have been shown to play essential roles in intercellular communications between cancer cells and the surrounding microenvironment owing to cancer development. EVs are small membrane-bound vesicles secreted by various cells containing proteins, lipids, mRNAs, and non-coding RNAs (microRNAs and long non-coding RNAs), which contribute to cancer cell development and progression. Here, we provide an overview of current research direction on EVs, especially biomolecules in EVs, and also point out the novel diagnostics, monitoring, predicting, and therapeutic aspects using EVs against cancer.

Safety and Prognostic Impacts of Ovarian Preservation during Radical Hysterectomy for Early-Stage Adenocarcinoma and Adenosquamous Cervical Cancer.

OBJECTIVE: To identify the incidence of ovarian metastasis and the impact of ovarian preservation on oncological outcomes for early-stage adenocarcinoma and adenosquamous cervical cancer. METHODS: 281 patients with stages IA2-IB1 adenocarcinoma and adenosquamous cervical cancer who underwent radical hysterectomy with pelvic lymphadenectomy (RHND) were included in the study. The incidence of ovarian metastasis was evaluated from 173 patients who underwent oophorectomy during RHND. Subgroup analysis was performed for patients less than 50 years (196 of 281 patients) who were classified into two groups, ovarian preservation and nonovarian preservation groups. 5-year recurrence-free survival (5-yr RFS) and 5-year overall survival (5-yr OS) were evaluated and compared between these groups. RESULTS: There was no evidence of ovarian metastasis, synchronous ovarian cancer, or ovarian recurrence during follow-up. In patients less than 50 years of age, there were no statistically significant differences in the 5-yr RFS (P = 0.363), or 5-yr OS (P = 0.974) between the ovarian preservation and nonovarian preservation groups. In Kaplan-Meier analysis, the ovarian preservation group seemed to have a slightly better OS in long-term follow-up (after 15 years); however, the difference was not statistically significant. CONCLUSIONS: Ovarian preservation was safe in adenocarcinoma and adenosquamous cervical cancer stages IA2-B1. However, the impact of ovarian preservation on oncological outcomes needs to be further investigated.

Impact of Obesity on Clinical Outcomes in Patients with Early-Stage Cervical Cancer after Radical Hysterectomy with Pelvic Node Dissection.

BACKGROUND: The aim of this study was to determine the prognostic role of obesity on oncological outcomes, surgical complications, and postoperative morbidity of patients with early-stage cervical cancer. METHODS: Between 2000 and 2016, we enrolled 500 patients with early-stage cervical cancer who underwent radical hysterectomy with pelvic node dissection (RHND) at Songklanagarind Hospital. For analysis, patients were divided based on their body mass index (BMI) into under-normal weight (<25 kg/m2), overweight (25-29.99 kg/m2), and obese (≥30 kg/m2) groups. RESULTS: The median age was 47 years, and the median BMI was 24.3 kg/m2 (25% quartile, 22.0 kg/m2; 75% quartile, 27.4 kg/m2). Patients in the obese and overweight groups were more likely to have comorbidities and adenocarcinoma than patients in the under-normal weight group. The median operative time (OT) was significantly longer in the obese and overweight groups than in the under-normal weight group. The 5-year recurrence-free survival (RFS) of the under-normal weight, overweight, and obese groups was 87.5, 86.2, and 97.6%, respectively, and the 5-year overall survival (OS) times were 95.8, 97.8, and 100%, respectively. There were no significant differences in RFS or OS among the 3 weight groups. Multivariate analysis did not identify BMI as a prognostic factor for RFS and OS. CONCLUSIONS: A high BMI was not associated with increased surgical complications or postoperative morbidity; furthermore, it was not associated with the prognosis of patients with early-stage cervical cancer after RHND. However, it was associated with adenocarcinoma and longer OT.

Impact of time interval between radical hysterectomy with pelvic node dissection and initial adjuvant therapy on oncological outcomes of early stage cervical cancer.

OBJECTIVE: To determine the impact of time interval (TI) from radical hysterectomy with pelvic node dissection (RHND) to adjuvant therapy on oncological outcomes in cervical cancer. METHODS: The study included 110 stage IA2-IB1 cervical cancer patients who underwent RHND and adjuvant therapy. The patients were divided into 2 groups based on the cut-off points of TI of 4 and 6 weeks, respectively. The associations of TI and clinicopathologic factors with oncological outcomes were evaluated using Cox proportional-hazards regression. RESULTS: The median TI was 4.5 weeks. There were no statistical differences in 5-year recurrence-free survival (RFS) (89.2% vs. 81.0%, and 83.2% vs. 100.0%) or 5-year overall survival (OS) rates (90.9% vs. 97.2%, and 93.2% vs. 100.0%) between patients according to TI (≤4 vs. >4, and ≤6 vs. >6 weeks, respectively). Deep stromal invasion (p=0.037), and parametrial involvement (PI) (p=0.002) were identified as independent prognostic factors for RFS, together with the interaction between TI and squamous cell carcinoma histology (p<0.001). In patients with squamous cell carcinoma, a TI longer than 4 weeks was significantly associated with a worse RFS (hazard ratio [HR]=15.8; 95% confidence interval [CI]=1.4-173.9; p=0.024). Univariate analysis showed that only tumor size (p=0.023), and PI (p=0.003) were significantly associated with OS. CONCLUSION: Delay in administering adjuvant therapy more than 4 weeks after RHND in early stage squamous cell cervical cancer results in poorer RFS.