A unique circulating microRNA pairs signature serves as a superior tool for early diagnosis of pan-cancer.

Cancer remains a major burden globally and the critical role of early diagnosis is self-evident. Although various miRNA-based signatures have been developed in past decades, clinical utilization is limited due to a lack of precise cutoff value. Here, we innovatively developed a signature based on pairwise expression of miRNAs (miRPs) for pan-cancer diagnosis using machine learning approach. We analyzed miRNA spectrum of 15832 patients, who were divided into training, validation, test, and external test sets, with 13 different cancers from 10 cohorts. Five different machine-learning (ML) algorithms (XGBoost, SVM, RandomForest, LASSO, and Logistic) were adopted for signature construction. The best ML algorithm and the optimal number of miRPs included were identified using area under the curve (AUC) and youden index in validation set. The AUC of the best model was compared to previously published 25 signatures. Overall, Random Forest approach including 31 miRPs (31-miRP) was developed, proving highly efficient in cancer diagnosis across different datasets and cancer types (AUC range: 0.980-1.000). Regarding diagnosis of cancers at early stage, 31-miRP also exhibited high capacities, with AUC ranging from 0.961 to 0.998. Moreover, 31-miRP exhibited advantages in differentiating cancers from normal tissues (AUC range: 0.976-0.998) as well as differentiating cancers from corresponding benign lesions. Encouragingly, comparing to previously published 25 different signatures, 31-miRP also demonstrated clear advantages. In conclusion, 31-miRP acts as a powerful model for cancer diagnosis, characterized by high specificity and sensitivity as well as a clear cutoff value, thereby holding potential as a reliable tool for cancer diagnosis at early stage.

Nanoreceptors promote mutant p53 protein degradation by mimicking selective autophagy receptors.

In some cancers mutant p53 promotes the occurrence, development, metastasis and drug resistance of tumours, with targeted protein degradation seen as an effective therapeutic strategy. However, a lack of specific autophagy receptors limits this. Here, we propose the synthesis of biomimetic nanoreceptors (NRs) that mimic selective autophagy receptors. The NRs have both a component for targeting the desired protein, mutant-p53-binding peptide, and a component for enhancing degradation, cationic lipid. The peptide can bind to mutant p53 while the cationic lipid simultaneously targets autophagosomes and elevates the levels of autophagosome formation, increasing mutant p53 degradation. The NRs are demonstrated in vitro and in a patient-derived xenograft ovarian cancer model in vivo. The work highlights a possible direction for treating diseases by protein degradation.

Photodynamic Modulation of Endoplasmic Reticulum and Mitochondria Network Boosted Cancer Immunotherapy.

Immunogenic cell death (ICD) represents a promising approach for enhancing tumor therapy efficacy by inducing antitumor immune response. However, current ICD inducers often have insufficient endoplasmic reticulum (ER) enrichment and ineffectiveness in tumor immune escape caused by ER-mitochondria interaction. In this study, a kind of photoactivatable probe, THTTPy-PTSA, which enables sequential targeting of the ER and mitochondria is developed. THTTPy-PTSA incorporates p-Toluenesulfonamide (PTSA) for ER targeting, and upon light irradiation, the tetrahydropyridine group undergoes a photo oxidative dehydrogenation reaction, transforming into a pyridinium group that acts as a mitochondria-targeting moiety. The results demonstrate that THTTPy-PTSA exhibits exceptional subcellular translocation from the ER to mitochondria upon light irradiation treatment, subsequently triggers a stronger ER stress response through a cascade-amplification effect. Importantly, the augmented ER stress leads to substantial therapeutic efficacy in a 4T1 tumor model by eliciting the release of numerous damage-associated molecular patterns, thereby inducing evident and widespread ICD, consequently enhancing the antitumor immune efficacy. Collectively, the findings emphasize the pivotal role of photodynamic modulation of the ER-mitochondria network, facilitated by THTTPy-PTSA with precise spatial and temporal regulation, in effectively bolstering the antitumor immune response. This innovative approach presents a promising alternative for addressing the challenges associated with cancer immunotherapy.

Noncoding RNAs: an emerging modulator of drug resistance in pancreatic cancer.

Pancreatic cancer is the eighth leading cause of cancer-related deaths worldwide. Chemotherapy including gemcitabine, 5-fluorouracil, adriamycin and cisplatin, immunotherapy with immune checkpoint inhibitors and targeted therapy have been demonstrated to significantly improve prognosis of pancreatic cancer patients with advanced diseases. However, most patients developed drug resistance to these therapeutic agents, which leading to shortened patient survival. The detailed molecular mechanisms contributing to pancreatic cancer drug resistance remain largely unclear. The growing evidences have shown that noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are involved in pancreatic cancer pathogenesis and development of drug resistance. In the present review, we systematically summarized the new insight on of various miRNAs, lncRNAs and circRNAs on drug resistance of pancreatic cancer. These results demonstrated that targeting the tumor-specific ncRNA may provide novel options for pancreatic cancer treatments.

SCARNA10 regulates p53 acetylation-dependent transcriptional activity.

The tumor suppressor p53 is involved in variety of cell progresses including cell cycle arrest, apoptosis, DNA repair, senescence, cell metabolism and ferroptosis. Here, we identified lncRNA SCARNA10 (Small Cajal Body-Specific RNA 10) as a novel cellular factor that interacts with the DNA binding domain (DBD) of p53. Upon binding the DBD of p53 and CREB-binding protein (CBP), SCARNA10 promotes the acetylation of p53, and activates p53-mediated transcriptional activation. Overexpress or knockdown SCARNA10 leads to up (or down)-regulation of p53-mediated transcriptional activation, whereas not affecting p53 protein levels. Moreover, SCARNA10 directly activates transcription by increasing the acetylation of p53 C-terminal domain (CTD) without affecting p53 phosphorylation at Ser15. These results indicate that SCARNA10 is a novel factor which regulates p53 acetylation-dependent transcriptional activity and tumor suppression.

[Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification].

OBJECTIVE: To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. METHODS: Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. RESULTS: The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). CONCLUSION: Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Pivotal roles of tumor-draining lymph nodes in the abscopal effects from combined immunotherapy and radiotherapy.

BACKGROUND: Currently, due to synergy enhancement of anti-tumor effects and potent stimulation of abscopal effects, combination therapy with irradiation and programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint inhibition (immuno-radiotherapy, iRT) has revolutionized the therapeutic guidelines. It has been demonstrated that tumor-draining lymph nodes (TDLN) are essential for effective antitumor immunity induced by radiotherapy, immunotherapy, or iRT. Given that the function of TDLN in iRT remains unclear, this study aimed to investigate the function and mechanism of TDLN in iRT-induced abscopal effects. METHODS: The function of TDLN was evaluated using unilateral or bilateral MC38 and B16F10 subcutaneous tumor models with or without indicated TDLN. The flow cytometry, multiple immunofluorescence analysis, and NanoString analysis were utilized to detect the composition and function of the immune cells in the primary and abscopal tumor microenvironment. Additionally, we tempted to interrogate the possible mechanisms via RNA-sequencing of tumor-infiltrating lymphocytes and TDLN. RESULTS: TDLN deficiency impaired the control of tumor growth by monotherapy. Bilateral TDLN removal rather than unilateral TDLN removal substantially curtailed iRT-stimulated anti-tumor and abscopal effects. Furthermore, in the absence of TDLN, the infiltration of CD45+ and CD8+ T cells was substantially reduced in both primary and abscopal tumors, and the anti-tumor function of CD8+ T cells was attenuated as well. Additionally, the polarization of tumor-associated macrophages in primary and abscopal tumors were found to be dependent on intact bilateral TDLN. RNA-sequencing data indicated that impaired infiltration and anti-tumor effects of immune cells partially attributed to the altered secretion of components from the tumor microenvironment. CONCLUSIONS: TDLN play a critical role in iRT by promoting the infiltration of CD8+ T cells and maintaining the M1/M2 macrophage ratio.

Aberrant ROS Served as an Acquired Vulnerability of Cisplatin-Resistant Lung Cancer.

Lung cancer has become a global health issue in recent decades. Approximately 80-85% of cases are non-small-cell lung cancer (NSCLC). Despite the high rate of resistance, cisplatin-base chemotherapy is still the main treatment for NSCLC patients. Thus, overcoming cisplatin resistance is urgently needed in NSCLC therapy. In this study, we identify NADPH metabolism and reactive oxygen species (ROS) levels as the main causes accounting for cisplatin resistance. Based on a small panel consisting of common chemotherapy drugs or compounds, APR-246 is proved to be an effective compound targeting cisplatin-resistant NSCLC cells. APR-246 specially inhibits proliferation and colony formation of cisplatin-resistant cells. In details, APR-246 can significantly cause G0/G1 accumulation and S phase arrest of cisplatin resistant cells and gives rise to severe mitochondria dysfunction as well as elevated apoptosis. Further study proves that it is the aberrant ROS levels as well as NRF2/SLC7A11/GSH axis dysfunction accounting for the specific antitumor effects of APR-246. Scavenging ROS with N-acetylcysteine (NAC) disrupts the inhibitory effect of APR-246 on cisplatin-resistant cells. Mechanistically, NRF2 is specifically degraded by the proteasome following its own ubiquitylation in APR-246-treated cisplatin-resistant cells, which in turn decreases NRF2/SLC7A11/GSH axis activity. Our study provides new insights into the biology driving cisplatin resistance of lung cancer and highlights APR-246 as a potential therapeutic reagent for overcoming cisplatin resistance.

An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation.

Here, we identified Dicer as a major cellular factor that recognizes the DNA binding domain (DBD) of p53 in a manner dependent on its acetylation status. Upon binding the unacetylated DBD, Dicer is recruited to the promoters of p53 target genes, where it represses p53-mediated transcriptional activation. Conversely, knockdown or knockout of endogenous Dicer leads to up-regulation of p53-mediated transcriptional activation without increasing its protein levels. Moreover, Dicer-mediated repression is independent of its intrinsic endoribonuclease activity; instead, Dicer directly represses transcription by recruiting the SUV39H1 histone methyltransferase. However, upon DNA damage, Dicer-mediated repression is abrogated by stress-induced acetylation at the DBD of p53. Notably, the inability of acetylation-defective p53-3KR in transcription is partially but significantly restored upon loss of Dicer expression. Our study reveals that Dicer acts as an unexpected acetylation "reader" for p53 and thus has important implications regarding the mechanism of acetylation-mediated regulation of p53 transcriptional program.

[Clinical efficacy of posterior percutaneous endoscopic cervical discectomy for single level cervical spondylopathy with intraspinal ossification].

OBJECTIVE: To evaluate the effectiveness and security of posterior percutaneous endoscopic cervical discectomy (PPECD) in the treatment of single level cervical spondylopathy with intraspinal ossification. METHODS: Twenty three patients with single level cervical spondylopathy with intraspinal ossification were treated by posterior percutaneous endoscopic cervical discectomy between August 2017 and July 2019. There were 16 males and 7 females, aged from 29 to 74 years old with an average of (50±13) years.The disease duration were 3 to 120 months with a median of 6 months. There were 9 cases of cervical spondylotic radiculopathy, 6 cases of cervical spondylotic myelopathy, and 8 cases of mixed cervical spondylopathy. According to the characteristics of ossification, 17 cases were osteophytes on the posterior edge of the vertebral body;3 cases were protrusion ossification;3 cases were posterior longitudinal ligament ossification. According to the position of ossification in spinal canal, 14 cases were medial and lateral type, 5 cases were central type, and 4 cases were mixed type. Posterior percutaneous cervical endoscopic cervical discectomy in patients performed by the same surgeon. Japanese Orthopaedic Association (JOA) score and visual analogue scale(VAS) were compared separately before and after operation. At 3 months after operation, clinical effect was assessed according to modified Macnab standard. RESULTS: All operations were successful. The operative time was 30 to 155 (69.1±27.2) min. The bedridden time was 2 to 3(3.0±0.9) h, length of postoperative hospitalization was 2 to 7(4.1± 1.5) d. Three dimensional CT reconstruction of the cervical spine at 3 days after operation showed that ossified tissue of 13 cases were completely removed, and 10 cases were left after operation, and the residual was located at the posterior edge and/or center of the upper vertebral body. VAS score at discharge from hospital was significantly lower than that before operation (t=9.35, P<0.001), and 21 cases had a score of 0 to 3. Postoperative JOA score was significantly higher (t=7.29, P<0.001). At 3 months after operation, according to modified Macnab standard to evaluate clinical effect, 18 cases got exellent results, 4 good and 1 fair, with an excellent and good rate of 95.6%(22/23). CONCLUSION: For an experienced surgeon, percutaneous posterior cervical endoscopic discectomy is safe and reliable in treating single level cervical spondylopathy with intraspinal ossification, and can obtain good clinical results.

[Analysis of clinical efficacy of C3 expanded half lamina excision combined with unilateral open-door laminoplasty for multiple segmental cervical spinal cord compression syndrome].

OBJECTIVE: To explore the clinical efficacy of C3 expanded half lamina excision combined with unilateral open door laminoplasty for multiple segmental cervical spinal cord compression syndrome. METHODS: The clinical data of 58 patients with multiple segmental cervical spinal cord compression syndrome underwent surgical treatment between September 2014 and May 2018 were retrospectively analyzed. There were 34 males and 24 females with a mean age of 64.4 years old (ranged from 46 to 78 years old). Among them, 28 cases received the surgery of C3 expanded half lamina excision combined with C4-C7 unilateral open-door laminoplasty (improvedgroup), and 30 cases received a single C3-C7 unilateral open-door laminoplasty (traditional group). Operation time, intraoperative blood loss, complications including C5 nerve root palsy and axial symptoms were compared between two groups. To evaluate the situation of the imaging indicators by measuring the space available for the spinal cord through cross sectional MRI of cervical spine at the narrowest segment of C3 (including intervertebral disc levels of C3, 4). Pre- and post-operative Japanese Orthopedic Association(JOA) score, Neck Disability Index(NDI) score, and improvement rate of neurological function, were recorded and analyzed between the two groups. RESULTS: All the patients were followed up for 12 to 18 months with an average of(14.5±1.8) months for improved group and (14.5±1.9) months for traditional group, and no significant difference was found between the two groups (P>0.05). There was no significant difference in intraoperative blood loss and C5 nerve root palsy between the two groups (P>0.05). The operation time (119±10) min vs (126±12) min and axial symptoms 7.1%(2/28) vs 26.6%(8/30) was significant difference between the two groups (P<0.05). Preoperative and postoperative space available for the spinal cord of C3 was (93.61±9.02) mm3 and (153.50±12.76) mm3 respectively, which was obtained obvious improvement in all patients(P<0.05). At the final follow up, JOA scores of improved group and traditional group were 14.36±1.70 and 14.03±1.82 respectively, and NDI scores were 10.36±2.55 and 12.47±3.46 respectively, there was significant difference between two groups (P<0.05). However, there was no significant difference between two groups for the improvement rate (68.36±0.12)%VS (65.01±0.12)%of neurological function(P>0.05). CONCLUSION: C3 expanded half lamina excision combined with unilateral open-door laminoplasty is an effective method to treat multiple segmental cervical spinal cord compression syndrome, for it can not only fully relieved spinal cord compression, but also achievedgood effect in preventing complications such as axial symptoms by reducing stripping of muscles from C2 spinous process.

Therapeutic effects of Co-Venenum Bufonis Oral Liquid on radiation-induced esophagitis in rats.

To investigate the effects of Co-Venenum Bufonis Oral Liquid (cVBOL) on radiation-induced esophagitis in rats. Irradiation (30 Gy) with X-RAD 225 x-ray was applied to induce esophagitis in 64 Wistar rats and treated by different methods. The body weight of rats either in RT group, cVBOL+RT, or EM+RT group was significantly decreased when compared with that in normal group (P<0.0001). After irradiation, histopathological studies, immunohistochemistry, and MRI scanning on esophagus were performed. Serum TNF-α,IL-6 and IL-10 were also determined by ELISA at 7, 14, 21 and 28 days after radiation treatment. The results demonstrated that radiation caused esophageal injury and thickening of esophageal tissue layers. The esophageal tissues after radiation treatment showed typical pathological changes of esophagitis. Radiation also caused esophagus edema. Treatment of cVBOL reduced the severity of histological esophageal lesion, decreased the expression of bFGF and TGF-ß1, and lowered serum levels of inflammatory cytokines including TNF-α, IL-6 and IL-10 over 28 days after radiation treatment. In conclusion, cVBOL treatment is effective to prevent radiation induced esophagitis and reduces radiation induced esophagitis may be mediated through its ant-inflammatory effects.

Noncoding RNAs in gastric cancer: implications for drug resistance.

Gastric cancer is the fourth most common malignancy and the third leading cause of cancer-related deaths worldwide. Advanced gastric cancer patients can notably benefit from chemotherapy including adriamycin, platinum drugs, 5-fluorouracil, vincristine, and paclitaxel as well as targeted therapy drugs. Nevertheless, primary drug resistance or acquisition drug resistance eventually lead to treatment failure and poor outcomes of the gastric cancer patients. The detailed mechanisms involved in gastric cancer drug resistance have been revealed. Interestingly, different noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), are critically involved in gastric cancer development. Multiple lines of evidences demonstrated that ncRNAs play a vital role in gastric cancer resistance to chemotherapy reagents and targeted therapy drugs. In this review, we systematically summarized the emerging role and detailed molecular mechanisms of ncRNAs impact drug resistance of gastric cancer. Additionally, we propose the potential clinical implications of ncRNAs as novel therapeutic targets and prognostic biomarkers for gastric cancer.

The emerging role of microRNAs and long noncoding RNAs in drug resistance of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the second most lethal human cancer. A portion of patients with advanced HCC can significantly benefit from treatments with sorafenib, adriamycin, 5-fluorouracil and platinum drugs. However, most HCC patients eventually develop drug resistance, resulting in a poor prognosis. The mechanisms involved in HCC drug resistance are complex and inconclusive. Human transcripts without protein-coding potential are known as noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), long noncoding RNAs (lncRNAs) and circular RNA (circRNA). Accumulated evidences demonstrate that several deregulated miRNAs and lncRNAs are important regulators in the development of HCC drug resistance which elucidates their potential clinical implications. In this review, we summarized the detailed mechanisms by which miRNAs and lncRNAs affect HCC drug resistance. Multiple tumor-specific miRNAs and lncRNAs may serve as novel therapeutic targets and prognostic biomarkers for HCC.

The emerging role of noncoding RNAs in colorectal cancer chemoresistance.

BACKGROUND: Colorectal cancer (CRC) is the third most prevalent cancer in the world and one of the most lethal human malignancies. Chemotherapy with 5-fluorouracil, platinum, hydroxycamptothecin, vincristine, methotrexate, irinotecan, paclitaxel and/or cetuximab has significantly improved the survival of CRC patients. However, most CRC patients eventually develop chemoresistance, resulting in a poor prognosis. The mechanisms involved in CRC chemoresistance are complex and, as yet, inconclusive. Noncoding RNAs (ncRNAs), such as small nucleolar RNAs (snoRNAs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), represent transcripts without protein-coding potential. Accumulating evidence indicates that multiple deregulated ncRNAs, including miRNAs and lncRNAs, play pivotal roles in the development of chemoresistance in CRC. This notion has potential clinical implications. CONCLUSIONS: In this review, we highlight the emerging roles and the regulatory mechanisms by which miRNAs and lncRNAs affect CRC chemoresistance. Tumor-specific miRNAs and lncRNAs may serve as novel therapeutic targets and prognostic biomarkers for CRC.

NEO212 induces mitochondrial apoptosis and impairs autophagy flux in ovarian cancer.

BACKGROUND: Temozolomide-perillyl alcohol conjugate (NEO212), a novel temozolomide (TMZ) analog, was previously reported to exert its anti-cancer effect in non-small cell lung cancer (NSCLC), and human nasopharyngeal carcinoma (NPC), etc.. In the current study, we intend to illuminate the potential anticancer property and the underly mechanisms of NEO212 in ovarian cancer cells. METHODS: The cytotoxicity of NEO212 was detected by MTT, colony formation analysis and xenograft model. The proteins involved in cell proliferation, DNA damage, autophagy and lysosomal function were detected by western blots; mitochondria, lysosome and autophagosome were visualized by TEM and/or immunofluorescence; Apoptosis, cell cycle analysis and mitochondrial transmembrane potential were detected by flow cytometry. TFEB translocation was detected by immunofluorescence and western blot. RESULTS: NEO212 has the potential anticancer property in ovarian cancer cells, as evidence from cell proliferation inhibition, G2/M arrest, DNA damage, xenograft, mitochondrial dysfunction and apoptosis. Importantly, we observed that although it induced significant accumulation of autophagosomes, NEO212 quenched GFP-LC3 degradation, down-regulated a series of lysosome related gene expression and blocked the autophagic flux, which significantly facilitated it induced apoptosis and was largely because it inhibited the nuclear translocation of transcription factor EB (EB). CONCLUSIONS: NEO212 inhibited TFEB translocation, and impaired the lysosomal function, implying NEO212 might avoid from autophagy mediated chemo-resistance, thus proposing NEO212 as a potential therapeutic candidate for ovarian cancer.

Morphine and ketamine treatment suppress the differentiation of T helper cells of patients with colorectal cancer in vitro.

There have been conflicting reports regarding the effects of anesthetic and analgesic drugs on immune function in patients with cancer. The aim of the present study was to investigate changes to T helper (Th) cell populations in patients with colorectal cancer (CRC) and to assess the effects of morphine and ketamine on the differentiation of Th cells harvested from patients with CRC in vitro. Peripheral blood samples were extracted from 20 patients with CRC and 20 healthy participants. Peripheral blood mononuclear cells were isolated and incubated in a solution containing phorbol-myristate-acetate (PMA) and ionomycin in the presence or absence of morphine or various ketamine concentrations (25, 50, and 100 µM). Samples were analyzed 4 h later. Th1 and Th2 cells were significantly increased by PMA and ionomycin stimulation; however, Th1 cells and the Th1/Th2 ratio in PMA and ionomycin treatments were significantly decreased in the CRC group compared with the control group. Following incubation with PMA and ionomycin, morphine significantly decreased Th1 cells and the Th1/Th2 ratio in the CRC group. Ketamine did not significantly affect levels of Th1 or Th2 cells or the Th1/Th2 ratio at a concentration of 25 µM; however, a significant increase in the Th1/Th2 ratio was observed at a concentration of 50 µM and, at 100 µM, a significant decrease in Th1 and Th2 cells and an increase in the Th1/Th2 ratio were observed. The present study suggests that CRC may shift the balance of Th1/Th2 towards Th2 by inducing an immunological response, morphine is able to suppress the differentiation of Th cells and decreases the Th1/Th2 ratio, and ketamine may affect the differentiation of Th cells in a dose-dependent manner.

Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation.

BACKGROUND: Temozolomide-perillyl alcohol conjugate (TMZ-POH), a novel Temozolomide (TMZ) analog developed based on the conjugation of TMZ and perillyl alcohol (POH), displayed strong anticancer potency in multiple cancer types. In this study, we aimed to clarify the relationship between TMZ-POH and autophagy, and explore the underlying mechanisms involved in. METHODS: The proteins involved in autophagy, mitochondrial fission, lysosomal function and membrane traffic were detected by western blots; Autophagosome, mitochondria and lysosome were visualized by transmission electron microscope (TEM) and immunostaining; Apoptosis analysis and fluorescence probe detection were applied by flow cytometry. RESULTS: TMZ-POH blocked mitophagy flux although the number of autophagosomes which colocalized with mitochondria in the cells was increased via inducing lysosomal dysfunction as evidence from impaired lysosomal acidification, maturation and hampered autophagosome- lysosome fusion, which largely depended on its downregulation on the small GTPase RAB7A via mevalonate pathway. More importantly, our data demonstrated TMZ-POH sensitized cancer cell to irradiation induced apoptosis. CONCLUSIONS: Temozolomide-perillyl alcohol conjugate impairs mitophagy flux by inducing lysosomal dysfunction in Non-Small Cell Lung Cancer (NSCLC) cells and sensitizes them to irradiation, thereby proposing TMZ-POH as a potential radiosensitizer.

Circulating tumor DNA measurement provides reliable mutation detection in mice with human lung cancer xenografts.

Genotype-directed targeted therapy has become one of the standard treatment options for non-small cell lung cancer (NSCLC). There have been numerous limitations associated with mutation analysis of tissue samples. Consequently, mutational profile analysis of circulating cell-free DNA (cfDNA) by highly sensitive droplet digital PCR (ddPCR) assay has been developed. Possibly due to differences in cfDNA concentrations, previous studies have shown numerous discrepancies in mutation detection consistency between tissue and cfDNA. In order to rigorously analyze the amount of cfDNA needed, we constructed 72 athymic nude mice xenografted with NCI-H1975 (harboring a EGFR T790M mutation) or NCI-H460 (harboring a KRAS Q61H mutation) human NSCLC. We thoroughly investigated the relationship between plasma cfDNA using Q-PCR targeting human long interspersed nuclear element-1 (LINE-1) retrotransposon and the mouse ACTB gene, and the accuracy of mutation detection by ddPCR at different times post-graft. Our results show that the concentration and fragmentation of human (tumor) derived cfDNA (hctDNA) were positively correlated with tumor weight, but not with mouse-derived cfDNA (mcfDNA). Quantification of cfDNA by Q-PCR depends on the amplified target length. Mutation copies in plasma of per milliliter were positively linked to tumor weight, hctDNA level and hctDNA/mcfDNA ratio, respectively. Furthermore, tumor weight, hctDNA level and ratio of hctDNA/mcfDNA were significantly higher in cfDNA mutation-positive mice than in negative mice. Also, our data indicate that when plasma hctDNA level and hctDNA/mcfDNA ratio reach a certain level in xenografted mice, plasma cfDNA mutation can be detected. In summary, the present study suggests that determination of ctDNA levels may be essential for reliable mutation detection by analysis of cfDNA.