ROS impairs tumor vasculature normalization through an endocytosis effect of caveolae on extracellular SPARC.

BACKGROUND: The accumulation of reactive oxygen species (ROS) in tumor microenvironment (TME) is an important player for tumorigenesis and progression. We aimed to explore the outcomes of ROS on tumor vessels and the potential regulated mechanisms. METHODS: Exogenous H2O2 was adopted to simulate the ROS setting. Immunofluorescence staining and ultrasonography were used to assess the vascular endothelial coverage and perfusions in the tumors inoculated with Lewis lung cancer (LLC) and melanoma (B16F10) cells of C57BL/6 mice, respectively. ELISA and western-blot were used to detect the expression of secreted acidic and cysteine-rich protein (SPARC) and Caveale-1 in human umbilical vein endothelial cells (HUVEC) extra- and intracellularly. Intracellular translocation of SPARC was observed using electron microscopy and immunofluorescence approaches. RESULT: Under the context of oxidative stress, the pericyte recruitment of neovascularization in mouse lung cancer and melanoma tissues would be aberrated, which subsequently led to the disruption of the tumor vascular architecture and perfusion dysfunction. In vitro, HUVEC extracellularly SPARC was down-regulated, whereas intracellularly it was up-regulated. By electron microscopy and immunofluorescence staining, we observed that SPARC might undergo transmembrane transport via caveale-1-mediated endocytosis. Finally, the binding of SPARC to phosphorylated-caveale-1 was also detected in B16F10 tissues. CONCLUSION: In the oxidative stress environment, neovascularization within the tumor occurs structural deterioration and decreased perfusion capacity. One of the main regulatory mechanisms is the migration of extracellular SPARC from the endothelium to intracellular compartments via Caveolin-1 carriers.

Clinical characteristics and novel strategies of immune checkpoint inhibitor rechallenge therapy for non-small cell lung cancer: a comprehensive review.

Treatment of non-small-cell lung cancer (NSCLC) has entered the immunotherapy era, marked by significant survival improvements due to the use of immune checkpoint inhibitors (ICIs). However, owing to factors, such as disease progression, long-term use, and side effects, some patients discontinue immunotherapy, resulting in limited subsequent treatment option and a negative impact on their survival and quality of life. We have collected relevant data which reveal that ICI rechallenge may be an effective clinical strategy. However, many factors affect the efficacy of rechallenge, including patient characteristics, initial treatment drugs, treatment duration, efficacy, toxicity, and side effects. Additionally, the side effects of rechallenge and mechanisms of reversing drug resistance play crucial roles. Identifying suitable candidates, optimizing treatment plans and duration, enhancing treatment efficacy, and minimizing toxicity and adverse effects in rechallenges are pressing clinical needs. Addressing these issues can provide guidance for the clinical use of immunotherapy rechallenges to better serve patients. This review focuses on the clinical considerations and strategies for immune therapy rechallenges in NSCLC.

Case report: A successful re-challenge report of GLS-010 (Zimberelimab), a novel fully humanized mAb to PD-1, in a case of recurrent endometrial cancer.

With the widespread use of immune checkpoint inhibitors (ICI), there is growing concern about reports of immune-related adverse events (irAE). In clinical practice, patients who experience severe toxicities by ICI-based therapies would require utmost caution in resuming ICI therapy because of the potential risk of serious irAEs caused by the reintroduction of immunotherapy. In this study, we report a case of recurrent endometrial cancer patient with PD-L1 positive as well as dMMR suffering from immunotherapy-associated myocarditis after first-line treatment with ICI combined with a multi-targeted anti-angiogenic agent. After symptomatic treatment, the patient was in complete remission from treatment toxicities. Subsequently, through MDT discussions, we selected a new PD-1 agent, zimberelimab, for rechallenge therapy, and the patient achieved a sustained disease remission without any treatment-related toxicities. To date, the manner and timing of the ICI re-challenge has been a subject of iterative deliberation. We believe that our experience could shed some light on ICI rechallenge therapy, and we look forward to more literatures to refine the ICI rechallenge scenarios.

Durable tracking anti-SARS-CoV-2 antibodies in cancer patients recovered from COVID-19.

Cancer patients are more susceptible to SARS-CoV-2 infection and generally have higher mortality rate. Anti-SARS-CoV-2 IgG is an important consideration for the patients in this COVID-19 pandemic. Recent researches suggested the rapid decay of anti-SARS-CoV-2 antibodies in the general population, but the decline rate of the antibodies in cancer patients was unknown. In this observational study, we reported the clinical features of the 53 cancer patients infected by SARS-CoV-2 from Wuhan, China and tracked the presence of anti-SARS-CoV-2 antibodies in the patients for more than 12 months. We found the duration (days) of anti-SARS-CoV-2 IgG in the patients was significant longer in chemotherapy (mean: 175; range: 75 to 315) and radiotherapy groups (mean: 168; range: 85 to 265) than in non-chemo- or radio-therapy group (mean: 58; range: 21 to 123) after their recovery from COVID-19. We also used single-cell RNA sequencing to track the immunologic changes in a representative patient recovered  from COVID-19 and found that CD8 + effective T cells, memory B cells and plasma cells were persistently activated in the patient undergoing chemotherapy. Together, our findings show that chemotherapy and radiotherapy might be beneficial to extend the duration of anti-SARS-CoV-2 IgG.

Targeting NEK2 impairs oncogenesis and radioresistance via inhibiting the Wnt1/ß-catenin signaling pathway in cervical cancer.

BACKGROUND: NEK2, a serine/threonine kinase involved in mitosis, has been found to function in chromosome instability, tumor progression and metastasis, but its role in cervical cancer radioresistance remains unknown. METHODS: We detected the protein levels of NEK2 in cervical carcinoma tissues and paired paracarcinoma tissues by immunohistochemistry. The roles of NEK2 in oncogenesis were examined using cell growth and colony formation assays, EdU assay, apoptosis assay as well as in vivo mouse model. γ-H2AX and Rad51 foci formation, neutral comet assay and clonogenic cell survival assay were applied to determine the radiosensitivity of cervical cancer cells. RNA-seq was performed to identify the downstream effector of NEK2. The gene expression levels were measured by Real-time PCR. RESULTS: We report that NEK2 protein level is overexpressed and correlated with the tumor stage and lymph node metastasis in cervical cancer tissues. Furthermore, we provided evidence that depletion of NEK2 impairs oncogenesis and enhances radiosensitivity in cervical cancer. Using RNA sequencing, we identify Wnt1 as a key downstream effector of NEK2. Knockdown of NEK2 downregulates the mRNA and protein levels of Wnt1, thereby inhibiting the activation of the Wnt/ß-catenin signaling pathway. More importantly, the observed consequences induced by NEK2 depletion in cervical cancer cells can be partially rescued by Wnt1 overexpression. CONCLUSIONS: Our results demonstrate that NEK2 activates the Wnt/ß-catenin signaling pathway via Wnt1 to drive oncogenesis and radioresistance in cervical cancer, indicating that NEK2 may be a promising target for the radiosensitization of cervical cancer.

PAK6 promotes cervical cancer progression through activation of the Wnt/ß-catenin signaling pathway.

p21-activated kinase 6 (PAK6), a member of the serine/threonine kinase family, has been reported to be involved in numerous types of cancers. The present study aimed to investigate the role of PAK6 in cervical cancer. In the present study, PAK6 expression was evaluated in tissue microarrays and cell lines by using immunohistochemistry and western blotting. The mRNA level of PAK6 was evaluated by reverse transcription quantitative PCR. The Wnt/ß-catenin signaling-related protein expression was detected by western blotting following short hairpin (sh)RNA-mediated PAK6 knockdown or PAK6 overexpression. Cell proliferation was determined using Cell Countink Kit-8. Migration, invasion and colony formation were further assessed following PAK6 knockdown or overexpression. Co-immunoprecipitation (Co-IP) and fluorescence colocalization microscopy were used to detect the interaction between PAK6 and GSK3ß. The results from tissue microarray revealed that the expression levels of PAK6 in cervical cancer tissues were upregulated. The downregulation of PAK6 expression levels using shRNA not only decreased cell growth and proliferation, but it also inhibited the migration and invasion of HeLa cells. Conversely, the overexpression of PAK6 promoted the proliferation, migration and invasion of HeLa cells. In addition, the expression levels of proteins involved in the Wnt/ß-catenin signaling pathway were modified in the PAK6 knockdown group, including downregulation of GSK3ß phosphorylation and Cyclin D1 protein, and upregulation of ß-catenin phosphorylation and E-cadherin. In contrast, following the overexpression of PAK6, the Wnt/ß-catenin signaling pathway was activated. Further investigation using fluorescence microscopy and Co-IP assays indicated that PAK6 may interact with GSK3ß. In conclusion, the findings of the present study suggested that PAK6 may serve a role in promoting cervical cancer through activating the Wnt/ß-catenin signaling pathway.

Patients with Cancer Appear More Vulnerable to SARS-CoV-2: A Multicenter Study during the COVID-19 Outbreak.

The novel COVID-19 outbreak has affected more than 200 countries and territories as of March 2020. Given that patients with cancer are generally more vulnerable to infections, systematic analysis of diverse cohorts of patients with cancer affected by COVID-19 is needed. We performed a multicenter study including 105 patients with cancer and 536 age-matched noncancer patients confirmed with COVID-19. Our results showed COVID-19 patients with cancer had higher risks in all severe outcomes. Patients with hematologic cancer, lung cancer, or with metastatic cancer (stage IV) had the highest frequency of severe events. Patients with nonmetastatic cancer experienced similar frequencies of severe conditions to those observed in patients without cancer. Patients who received surgery had higher risks of having severe events, whereas patients who underwent only radiotherapy did not demonstrate significant differences in severe events when compared with patients without cancer. These findings indicate that patients with cancer appear more vulnerable to SARS-CoV-2 outbreak. SIGNIFICANCE: Because this is the first large cohort study on this topic, our report will provide much-needed information that will benefit patients with cancer globally. As such, we believe it is extremely important that our study be disseminated widely to alert clinicians and patients.This article is highlighted in the In This Issue feature, p. 747.

Low heme oxygenase-1 expression promotes gastric cancer cell apoptosis, inhibits proliferation and invasion, and correlates with increased overall survival in gastric cancer patients.

Heme oxygenase-1 (HO-1) plays a key role in anti-oxidation, anti-apoptosis, and anti-proliferation in various types of cancers. However, the relationship between HO-1 expression and gastric cancer development remains largely unknown. In this study, the protein expression of HO-1 in human gastric cancer was measured by immunohistochemistry on paraffin sections of 89 paired gastric carcinoma tissues and adjacent non-cancer tissues. The correlation of HO-1 expression with 5-year overall survival rate was estimated. The effects of decreased HO-1 expression by two strands of small interfered RNAs (siRNAs) on cell apoptosis, proliferation, and invasion of gastric cancer cell lines were examined by flow cytometry, the MTT assay, and the cell migration assay, respectively. High expression of HO-1 was detected in 11.2% (10/89) of gastric carcinoma tissues, compared with 1.1% (1/89) in matched adjacent normal tissues, and correlated with a decreased survival rate in gastric cancer patients. There were no significant correlations between HO-1 expression and clinical characteristics. Downregulation of HO-1 expression using two strands of siRNAs promoted apoptosis and inhibited the proliferation and invasion of two gastric cancer cell lines, SGC7901 and MKN-28 cells. This study demonstrated that HO-1 plays a vital role in the development of gastric cancer and may serve as a therapeutic target of this type of cancer.

Small-arc volumetric-modulated arc therapy: A new approach that is superior to fixed-field IMRT in optimizing dosimetric and treatment-relevant parameters for patients undergoing whole-breast irradiation following breast-conserving surgery.

Volumetric-modulated arc therapy (VMAT) is considered to deliver a better dose distribution and to shorten treatment time. There is a lack of research regarding breast irradiation after breast-conserving surgery (BCS) using VMAT with prone positioning. We developed a new small-arc VMAT methodology and compared it to conventional (fixed-field) intensity-modulated radiation therapy (IMRT) in the dosimetric and treatment relevant parameters for breast cancer patients in the prone position.Ten early-stage breast cancer patients were included in this exploratory study. All patients underwent computed tomography (CT) simulation scan in the prone position and for each patient, IMRT and VMAT plans were generated using the Monaco planning system. Two symmetrical partial arcs were applied in the VMAT plans. The angle ranges of the 2 arcs were set to approximately 60° to 100° and 220° to 260°, with small adjustments to maximize target coverage, while minimizing lung and heart exposure. The IMRT plans used 4 fixed fields. Prescribed doses were 50 Gy in 25 fractions. The target coverage, homogeneity, conformity, dose to organs at risk (OAR), treatment time, and monitor units (MU) were evaluated.Higher median conformal index (CI) and lower homogeneity index (HI) of the planning target volume (PTV) were respectively observed in VMAT and plans group (CI, 95% vs 91%; HI, 0.09 vs 0.12; P < 0.001). The volumes of ipsilateral lung receiving 30, 20, 10, and 5 Gy were lower for VMAT (P < 0.01), being 10%, 14.9%, 25.9%, and 44.9%, respectively, compared to 11.79%, 17.32%, 30.27%, and 50.58% for the IMRT plans. The mean lung dose was also reduced from 10.6 ±â€Š1.8 to 9.6 ±â€Š1.4 Gy (P = 0.001). The volumes of the heart receiving 30 and 40 Gy were similar for the 2 methods. In addition, the median treatment time (161 vs 412 seconds; P < 0.001) and the mean MU (713 vs 878; P < 0.001) were lower for VMAT.Small-arc VMAT plan improved CI and HI for the target, spared the dose of lung, and reduced treatment time and MU, compared to IMRT. It is a more promising irradiation technique for post-BCS radiotherapy.

Evaluation of HO-1 expression, cellular ROS production, cellular proliferation and cellular apoptosis in human esophageal squamous cell carcinoma tumors and cell lines.

Patients with esophageal squamous cell carcinoma (ESCC) have a poor prognosis. However, the related mechanisms are unclear, thus we investigated the expression of HO-1 in ESCC tissue and explored possible mechanisms of tumor progression. Expression of HO-1 was examined by immunohistochemistry in 143 ESCC tumors. The correlation of HO-1 with clinicopathological characteristics was also examined. Two human ESCC cell lines, TE-13 and Eca109 were studied. Silencing of cell line HO-1 by specific small interfering RNA (siRNA) was evaluated using real-time quantitative PCR. Cell line viability, apoptosis and intracellular levels of reactive oxygen species (ROS) after transfection were determined using MTT and flow cytometry, respectively. HO-1, Bax, Bcl-2 and A-caspase-3/-9 expression was evaluated using western blot analyses. We found that HO-1 was expressed in 58 of 143 ESCC tumors, mainly in the cytoplasm. There was a significant association between HO-1 expression and tumor grade (P<0.001). Knockdown of HO-1 expression in cell lines was associated with significantly decreased cellular proliferation (P<0.05) and a higher rate of apoptosis (P<0.001) 48 h after treatment. Treatment of the cell lines with the ROS inhibitor N-acetylcysteine abrogated this effect. Knockdown of HO-1 was associated with increased A-caspase-3 and -9 expression, but no change in Bax or Bcl-2 expression or Bax/Bcl-2 ratio was observed. Thus, the present study identified that ESCC tumors frequently expressed HO-1. Knockdown of HO-1 promoted apoptosis through activation of a ROS-mediated caspase apoptosis pathway.

Endothelial NO synthase and reactive oxygen species mediated effect of simvastatin on vessel structure and function: pleiotropic and dose-dependent effect on tumor vascular stabilization.

Statins have been confirmed with protective effect to microvessels in diabetic retinopathy, implicated as reducing vascular permeability, maintaining endothelial junction integrity and improving blood perfusion, which are surrogate markers of vascular 'normalization', but no data are currently available on efficacy of statins on tumor endothelial functions. Since statins have been shown to exhibit a biphasic dose-related response on biological behaviour of microvessels, we sought to determine whether statins with bipolar concentrations can ameliorate vessel dysfunction and then increase efficiency of chemotherapeutics in Lewis lung carcinoma and B16F10 melanoma models. Our in vitro study showed that simvastatin reduces hypoxia-induced endothelium leakage in human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. In tumor-bearing mice, low-dose simvastatin (0.2 mg/kg) induced upregulation of endothelial NO synthase (eNOS) skewed vessels to a pericyte-coated and stable pattern, while high-dose simvastatin (10 mg/kg) remarkably deceased reactive oxygen species (ROS)-induced hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) expression, attenuating VEGF-drived tumor vessel hyperpermeability. These changes ultimately improved intratumoral perfusion and decreased tumor hypoxia. Administration of cisplatin and cyclophosphamide to the simvastatin-treated mice resulted in slower tumor growth. Collectively, simvastatin may promote tumor vessel normalization and show clinical benefit when used in combination with chemotherapeutics.

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma.

BACKGROUND: Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a preliminary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma. METHODS: We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation. RESULTS: Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01). CONCLUSION: Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.

Effects of Oxymatrine on the apoptosis of human esophageal carcinoma Eca109 cell line and its mechanism.

The effects of Oxymatrine (Oxy) on the proliferation and apoptosis of human esophageal carcinoma Eca109 cell line and the mechanism were investigated. The human esophageal carcinoma Eca109 cells were cultured in vitro. The Oxy-induced apoptosis of Eca 109 cells was assayed by using flow cytometry. The expressions of p-ERK(1/2), Cyclin D1, p21(waf/cip1), Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca109 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK(1/2), Cyclin D1 and Bcl-2, but up-regulated the expression of p21(waf/cip1) and Bax, and the ratio of Bax/Bcl-2 was increased. It was suggested the Oxy could induce the apoptosis of Eca109 cells, which might be related to the upregulation of p21(waf/cip1) and the downregulation of p-ERK(1/2), Cyclin D1 and p21(waf/cip1). The possible pathway may be related to Bcl-2/Bax.

Effects of Oxymatrine on the Apoptosis of Human Esophageal Carcinoma Eca109 Cell Line and Its Mechanism / 华中科技大学学报（医学）（英德文版）

The effects of Oxymatrine (Oxy) on the proliferation and apoptosis of human esophageal carcinoma Eca109 cell line and the mechanism were investigated. The human esophageal carcinoma Eca109 cells were cultured in vitro. The Oxy-induced apoptosis of Eca109 cells was assayed by using flow cytometry. The expressions of p-ERK1/2, Cyclin D1, p21waf/cip1, Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca109 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK1/2, cyclin D1 and Bc1-2, but up-regulated the expression of p21waf/cip1 and Bax, and the ratio of Bax/Bcl-2 was increased It was suggested the Oxy could induce the apoptosis of Eca109-cells, which might be related to the upregulation of p21waf/cip1 and the downregulation of p-ERK1/2 Cyclin D1 and p21waf/cip1. The possible pathway may be related to Bcl-2/Bax.