Pathway Blocker Research Articles

RETRACTED ARTICLE: Large tumor suppressor kinase 2 overexpression attenuates 5-FU-resistance in colorectal cancer via activating the JNK-MIEF1-mitochondrial division pathway

Background5-Fluorouracil (5-FU) is a standard treatment for colorectal cancer, but most patients develop 5-FU resistance. Here, we conducted experiments to identify an effective approach to augment 5-FU-based treatment in colorectal cancer in vitro.MethodsSW480 cells were in the present study and treated with 5-FU. Besides, LATS2 adenovirus vectors were infected into SW480 cells. Western blotting, immunofluorescence and ELISA were used to evaluate cell death and mitochondrial function. Pathway blocker was used to verify the role of MAPK-JNK pathway in SW480 cell death.ResultsAn obvious drop in large tumor suppressor kinase 2 (LATS2) expression was observed in SW480 cells after treatment with 5-FU. In addition, upregulation of LATS2 expression through infection with LATS2 adenovirus further increased the reduction of SW480 cell viability induced by 5-FU. Functional exploration showed that 5-FU treatment suppressed mitochondrial membrane potential, enhanced cyt-c release into the nucleus, induced an oxidative injury environment by promoting ROS production, and eventually upregulated Bax-related mitochondrial apoptosis. Besides, LATS2 overexpression in combination with 5-FU treatment further perturbed mitochondrial homeostasis, and this effect was achieved by elevating mitochondrial division. Mechanistically, LATS2 overexpression and 5-FU co-treatment amplified mitochondrial division by upregulating MIEF1 expression in a manner dependent on MAPK-JNK axis. Knockdown of MIEF1 using an siRNA-mediated loss of function assay and/or inhibition of the MAPK-JNK pathway using the specific inhibitor SP600125 abolished LATS2/5-FU-mediated deleterious effects on mitochondrial performance and SW480 cell viability.ConclusionsIn light of the above findings, LATS2 downregulation could be a potential mechanism of low response to 5-FU treatment. Overexpression of LATS2 to further disrupt mitochondrial function via the JNK-MIEF1 signalling pathway might be a method to optimize 5-FU-based chemotherapy.

Liraglutide promotes apoptosis of HepG2 cells by activating JNK signaling pathway.

To study the effect and mechanism of liraglutide on the apoptosis of human hepatocellular carcinoma HepG2 cells. HepG2 cell was treated with different concentrations of liraglutide at 0, 1, 10, 100, and 1000 nmol/L. The effect of liraglutide on HepG2 proliferation was detected by Cell Counting Kit-8 (CCK-8) method; the effect of liraglutide on the protein expression of c-Jun NH2-terminal Kinase (JNK) and phosphorylated JNK (p-JNK) was detected by Western blot; the degree of HepG2 apoptosis was observed by flow cytometry, and JNK pathway blocker SP600125 was used to further confirm that liraglutide promoted HepG2 apoptosis by regulating JNK signaling pathway. The proliferation inhibition rate of HepG2 cells increased with time and the increase in the concentration of liraglutide. The proliferation inhibition rate was the strongest when cultured for 48 h, and the IC50 (half maximal inhibitory concentration) was about 100 nmol/L of liraglutide. 100 nmol/L liraglutide was selected as the intervention condition for subsequent use of SP600165. The apoptosis rate of HepG2 cells increased with the increase of liraglutide's concentration. The apoptosis rate of HepG2 cells at blocker SP600125+100 nmol/L liraglutide was significantly lower than that at 100 nmol/L liraglutide alone (p<0.05). There was no significant difference in the expression of JNK protein in HepG2 cells at different concentrations of liraglutide (p>0.05). There was no significant difference in the expression of JNK protein in HepG2 cells using JNK pathway blocker SP600125 (p>0.05), while using JNK pathway blocker SP600125 significantly up-regulated the expression of p-JNK protein in HepG2 cells than 100 nmol/L of liraglutide alone (p<0.05). Liraglutide can promote the apoptosis of hepatocellular carcinoma HepG2 cells in a dose-dependent manner, and its mechanism may act by promoting the activation of the JNK signaling pathway.

Yap-Hippo promotes A549 lung cancer cell death via modulating MIEF1-related mitochondrial stress and activating JNK pathway

Although the role of Yes-associated protein (Yap) has been described in the progression of lung cancer, the downstream effector of the Yap-Hippo pathway has not been identified. Accordingly, the aim of our study is to explore whether Yap modulates the activity of lung cancer by controlling mitochondrial elongation factor 1 (MIEF1)-related mitochondrial stress in a manner dependent on the JNK pathway. Cell viability was determined via MTT, LDH release and immunofluorescence assays. ATP production, the mitochondrial membrane potential, and caspase-9 activity were investigated to assess mitochondrial function. siRNA transfection and pathway blockers were used to observe the roles of MIEF1 and JNK in Yap-modulated cell viability in lung cancer cells in vitro. Yap deletion reduced cell viability in A549 and H358 lung cancer cells. At the molecular level, Yap deletion promoted mitochondrial dysfunction, as evidenced by the decreased mitochondrial potential, increased mitochondrial oxidative stress, augmented mitochondrial pro-apoptotic factor leakage and elevated caspase-9 activity. In addition, we found that Yap modulated mitochondrial stress via MIEF1 and that loss of MIEF1 abolished the regulatory actions of Yap on mitochondrial stress and cell viability. Besides, we provided evidence to support the necessary role of JNK in Yap-mediated MIEF1 upregulation. Inhibition of JNK abolished the promotive effect of Yap deletion on MIEF1 activation. Taken together, our results identified the JNK-MIEF1 pathway and mitochondrial stress as downstream effectors of Yap in lung cancer. This finding suggests a novel approach for the treatment of lung cancer in clinical practice.

Genetic ablation of TAZ induces HepG2 liver cancer cell apoptosis through activating the CaMKII/MIEF1 signaling pathway.

Background and objectiveTranscriptional coactivator with PDZ-binding motif (TAZ) has been found to be associated with tumor progression. Mitochondrial homeostasis regulates cancer cell viability and metastasis. However, the roles of TAZ and mitochondrial homeostasis in liver cancer viability have not been explored. The aim of our study was to investigate the influence of TAZ on HepG2 liver cancer cell apoptosis.Materials and methodsHepG2 liver cancer cell was used in the present study, and shRNA against TAZ was transfected into HepG2 cell to knockdown TAZ expression. Mitochondrial function was analyzed using Western blotting, immunofluorescence assay, and flow cytometry. Pathway blocker was used to confirm the role of CaMKII pathway in TAZ-mediated cancer cell death.ResultsOur results indicated that TAZ deletion induced death in HepG2 cell via apoptosis. Biological analysis demonstrated that mitochondrial stress, including mitochondrial bioenergetics disorder, mitochondrial oxidative stress, and mitochondrial apoptosis, were activated by TAZ deletion. Furthermore, we found that TAZ affected mitochondrial stress by triggering mitochondrial elongation factor 1 (MIEF1)-related mitochondrial dysfunction. The loss of MIEF1 sustained mitochondrial function and promoted cancer cell survival. Molecular investigation illustrated that TAZ regulated MIEF1 expression via the CaMKII signaling pathway. Blockade of the CaMKII pathway prevented TAZ-mediated MIEF1 upregulation and improved cancer cell survival.ConclusionTaken together, our results highlight the key role of TAZ as a master regulator of HepG2 liver cancer cell viability via the modulation of MIEF1-related mitochondrial stress and the CaMKII signaling pathway. These findings define TAZ and MIEF1-related mitochondrial dysfunction as tumor suppressors that act by promoting cancer apoptosis via the CaMKII signaling pathway, with potential implications for new approaches to liver cancer therapy.

Mst1-Hippo pathway triggers breast cancer apoptosis via inducing mitochondrial fragmentation in a manner dependent on JNK-Drp1 axis.

Background and objectiveMst1-Hippo pathway and mitochondrial fragmentation participate in the progression of several types of cancers. However, their roles in breast cancer requires investigation. The aim of our study is to determine whether Mst1 overexpression regulates the viability of breast cancer cells via modulating mitochondrial fragmentation.Materials and methodsTUNEL staining, MTT assay and Western blotting were used to detect cancer cell death. Adenovirus-loaded Mst1 was transfected into cells to overexpress Mst1. Mitochondrial fragmentation was observed via immunofluorescence staining and Western blotting. Pathway blocker was used to detect whether Mst1 modulated cell death and mitochondrial fragmentation via JNK signaling pathway.ResultsOur data showed that Mst1 overexpression promoted breast cancer cell death in a manner dependent on mitochondrial apoptosis. Mitochondrial oxidative stress, energy metabolism disorder, mitochondrial cyt-c liberation and mitochondrial apoptosis activation were observed after Mst1 overexpression. Furthermore, we demonstrated that Mst1 overexpression activated mitochondrial stress via triggering Drp1-related mitochondrial fragmentation, and that inhibition of Drp1-related mitochondrial fragmentation abrogated the proapoptotic effect of Mst1 overexpression on breast cancer cells. To this end, we found that Mst1 modulated Drp1 expression via the JNK signaling pathway, and that blockade of the JNK pathway attenuated mitochondrial stress and repressed apoptosis in Mst1-overexpressed cells.ConclusionAltogether, our results identified a tumor suppressive role for Mst1 overexpression in breast cancer via activation of the JNK–Drp1 axis and subsequent initiation of fatal mitochondrial fragmentation. Given these findings, strategies to enhance Mst1 activity and elevate the JNK–Drp1–mitochondrial fragmentation cascade have clinical benefits for patients with breast cancer.

C3 glomerulopathy - understanding a rare complement-driven renal disease.

The C3 glomerulopathies are a group of rare kidney diseases characterized by complement dysregulation occurring in the fluid phase and in the glomerular microenvironment, which results in prominent complement C3 deposition in kidney biopsy samples. The two major subgroups of C3 glomerulopathy - dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) - have overlapping clinical and pathological features suggestive of a disease continuum. Dysregulation of the complement alternative pathway is fundamental to the manifestations of C3glomerulopathy, although terminal pathway dysregulation is also common. Disease is driven by acquired factors in most patients - namely, autoantibodies that target the C3 or C5 convertases. These autoantibodies drive complement dysregulation by increasing the half-life of these vital but normally short-lived enzymes. Genetic variation in complement-related genes is a less frequent cause. No disease-specific treatments are available, although immunosuppressive agents and terminal complement pathway blockers are helpful in some patients. Unfortunately, no treatment is universally effective or curative. In aggregate, the limited data on renal transplantation point to a high risk of disease recurrence (both DDD and C3GN) in allograft recipients. Clinical trials are underway to test the efficacy of several first-generation drugs that target the alternative complement pathway.

Sirtuin 3 inhibition induces mitochondrial stress in tongue cancer by targeting mitochondrial fission and the JNK-Fis1 biological axis.

Sirtuin 3 (Sirt3)-modified mitochondrial fission participates in the progression of several types of cancers. However, its role in tongue cancer requires investigation. The aim of our study is to determine whether Sirt3 knockdown regulates the viability of tongue cancer cells via modulating mitochondrial fission. Two types of tongue cancer cells were used in the present study, and siRNA was transfected into the cells to suppress Sirt3 expression. Mitochondrial function and cell apoptosis were determined via immunofluorescence, Western blotting, ELISA, and qPCR assays. A pathway blocker was applied to verify the role of the JNK-Fis1 signaling pathway in regulation of mitochondrial fission. The present study showed that loss of Sirt3 promoted tongue cancer cell death in a manner dependent on mitochondrial apoptosis. Mitochondrial oxidative stress, energy metabolism disorder, mitochondrial cyt-c liberation, and mitochondrial apoptosis activation were observed after Sirt3 silencing. Furthermore, we demonstrated that Sirt3 knockdown activated mitochondrial stress via triggering Fis1-related mitochondrial fission and that inhibition of Fis1-related mitochondrial fission abrogated the pro-apoptotic effect of Sirt3 knockdown on tongue cancer cells. To this end, we found that Sirt3 modulated Fis1 expression via the c-Jun N-terminal kinases (JNK) signaling pathway and that blockade of the JNK pathway attenuated mitochondrial stress and repressed apoptosis in Sirt3 knockdown cells. Altogether, our results identified a tumor-suppressive role for Sirt3 deficiency in tongue cancer via activation of the JNK-Fis1 axis and subsequent initiation of fatal mitochondrial fission. Given these findings, strategies to repress Sirt3 activity and enhance the JNK-Fis1-mitochondrial fission cascade have clinical benefits for patients with tongue cancer.

Epigenetical Targeting of the FOXP3 Gene by S-Adenosylmethionine Diminishes the Suppressive Capacity of Regulatory T Cells Ex Vivo and Alters the Expression Profiles.

Regulatory T cells (Treg cells), a subgroup of CD4 lymphocytes, play a crucial role in serving as an immune suppressor and in maintaining peripheral tolerance. As the accumulation of Treg cells in the tumor microenvironment is significantly associated with a decreased survival time of patients, they are considered as an important therapeutic target in the immunotherapy of human cancers. These cells are either derived from the thymus, which are called (CD4CD25CD127) natural Treg cells (nTreg cells), or they are generated from CD4CD25 naive T cells by transforming growth factor-beta 1 and interleukin 2 (IL-2) in the periphery, which are called induced Treg cells (iTreg cells). Although iTreg cells are unstable, nTreg cells stably express forkhead box P3 (FOXP3) protein. Moreover, nTreg cells can be classified as memory (CD45RA) and naive (CD45RA) Treg cells, and this classification is based on the expression of CD45RA. FOXP3, which is a master regulator transcription factor, is essential for the functions of Treg cells, and it is mainly controlled by epigenetic mechanisms. The cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway is also reported to contribute to the regulatory functions of tumor-infiltrating Treg cells. As a new approach, we investigated whether S-adenosylmethionine (SAM), a substrate of DNA methyltransferase, attenuates the immune-suppressive capacity of the naive subtype of nTreg cells (CD4CD25CD127CD45RA). Moreover, we examined the effects of PGE2/COX2 pathway blockers on the suppressive capacity of Treg cells. We found that SAM diminished the suppression competency of Treg cells by decreasing the FOXP3 mRNA and protein levels in a dose-dependent manner. SAM increased the DNA methylation of FOXP3 at the first intron site. In addition, SAM decreased the mRNA and protein levels of the IL-10 cytokine, which has suppressive roles in the immune system. Moreover, mRNA levels of interferon gamma (IFNG) were found to be increased. COX2 inhibition and blockage of PGE2 receptors also reduced the protein and mRNA levels of IL-10, but they did not exhibit any significant effect on Treg cells' suppression in the coculture system. Our results show that SAM might be considered and investigated as a promising agent for immunotherapy in the future.

RETRACTED: Cytokine augments the sorafenib-induced apoptosis in Huh7 liver cancer cellby inducing mitochondrial fragmentation and activating MAPK-JNKsignalling pathway

Sorafenib is a standard targeted drug used to treat hepatocellular carcinoma (HCC). Notably, cytokine has been found to further enhance the therapeutic effectiveness of the targeted drug. Thereby, the aim of this study is to verify whether cytokine IL-2 could increase the anti-cancer effects of sorafenib on liver cancer in vitro. Huh7 cells were used in the present study and the cell apoptosis and migration were determined in response to sorafenib treatment. Then, siRNA and pathway blocker were used to determine the molecular mechanisms by which IL-2 enhance the therapeutic effectiveness of Huh7 liver cancer cell in vitro. The data in our study illustrated that sorafenib treatment induced apoptosis in Huh7 liver cancer cell in vitro, an effect that was accompanied with a drop in cell proliferation and migration. Biological investigation demonstrated that IL-2 supplementation further augmented the pro-apoptotic effects of sorafenib in vitro. At the molecular levels, the combination of IL-2 and sorafenib impaired mitochondrial respiratory function, reduced mitochondrial potential, promoted mitochondrial ROS overloading and activated mitochondrial apoptotic pathway. Meanwhile, we found that IL-2 supplementation induced mitochondrial stress via activating mitochondrial fragmentation in a manner dependent on MAPK-JNK signalling pathway and TAZ protein. Blockade of the JNK signalling pathway and/or knockdown of TAZ could abrogate the inhibitor effects of IL-2/sorafenib on liver cancer survival, growth and mobility. Collectively, these data indicated that IL-2 supplementation could further augment the anti-cancer effectiveness of sorafenib via activating mitochondrial fragmentation in a manner dependent on MAPK-JNK signalling pathway and TAZ protein. This finding identifies mitochondrial stress and the JNK-Hippo pathway as the potential targets to treat liver cancer.

(2R,6R)-Hydroxynorketamine promotes dendrite outgrowth in human inducible pluripotent stem cell-derived neurons through AMPA receptor with timing and exposure compatible with ketamine infusion pharmacokinetics in humans.

The mechanisms underlying the prolonged antidepressant effects after a single infusion of ketamine are only partially understood. Ketamine's half-life of ∼2 h cannot explain antidepressant effects that last for 1 week, suggesting the triggering of long-lasting neuroplasticity. Recent human pharmacokinetics (PK) data indicate that a ketamine metabolite, (2R,6R)-hydroxynorketamine (HNK), persists in the high submicromolar range for additional 6-12 h. As in rodents HNK can induce dendrite outgrowth through AMPA receptor-mediated mechanisms, in this work, we aimed to show that HNK produces similar effects in human neurons at concentrations and exposure time compatible with human PK after ketamine infusion. Human dopaminergic neurons were differentiated in-vitro from inducible pluripotent stem cells obtained from healthy donors. Exposure to submicromolar HNK for 6 h produced dendrite outgrowth when measured 3 days after exposure. This neuroplasticity was similar to that obtained with exposure to low micromolar concentrations of ketamine for 1 or 6 h. HNK and ketamine effects were blocked by pretreatment with the AMPA receptor antagonists NBQX and GYKI 52466, and by the mammalian target of rapamycin pathway blocker rapamycin. It is reasonable to conclude that the mechanistic similarity between ketamine and HNK and their diachronic brain exposure owing to the different plasma PK observed after single therapeutic ketamine infusion should contribute to the final sustained antidepressant action.

RETRACTED ARTICLE: Excessive mitochondrial fragmentation triggered by erlotinib promotes pancreatic cancer PANC-1 cell apoptosis via activating the mROS-HtrA2/Omi pathways

BackgroundMitochondrial fragmentation drastically regulates the viability of pancreatic cancer through a poorly understood mechanism. The present study used erlotinib to activate mitochondrial fragmentation and then investigated the downstream events that occurred in response to mitochondrial fragmentation.MethodsCell viability and apoptosis were determined via MTT assay, TUNEL staining and ELISA. Mitochondrial fragmentation was measured via an immunofluorescence assay and qPCR. siRNA transfection and pathway blockers were used to perform the loss-of-function assays.ResultsThe results of our study demonstrated that erlotinib treatment mediated cell apoptosis in the PANC-1 pancreatic cancer cell line via evoking mitochondrial fragmentation. Mechanistically, erlotinib application increased mitochondrial fission and reduced mitochondrial fusion, triggering mitochondrial fragmentation. Subsequently, mitochondrial fragmentation caused the overproduction of mitochondrial ROS (mROS). Interestingly, excessive mROS induced cardiolipin oxidation and mPTP opening, finally facilitating HtrA2/Omi liberation from the mitochondria into the cytoplasm, where HtrA2/Omi activated caspase-9-dependent cell apoptosis. Notably, neutralization of mROS or knockdown of HtrA2/Omi attenuated erlotinib-mediated mitochondrial fragmentation and favored cancer cell survival.ConclusionsTogether, our results identified the mROS-HtrA2/Omi axis as a novel signaling pathway that is activated by mitochondrial fragmentation and that promotes PANC-1 pancreatic cancer cell mitochondrial apoptosis in the presence of erlotinib.

Norepinephrine enhances cell viability and invasion, and inhibits apoptosis of pancreatic cancer cells in a Notch‑1‑dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive types of cancer, which is associated with a poor prognosis due to complexities in prevention, early diagnosis and effective treatment. The lack of understanding regarding its induction and specific pro‑cancer mechanisms may contribute to its poor prognosis. The Notch‑1 pathway is widely considered to be a critical tumor‑promoting factor in PDAC. Previous studies have indicated that chronic psychological stress may promote the development of PDAC partially via the main downstream stress hormone, norepinephrine (NE); however, to the best of our knowledge, the role of the Notch‑1 pathway in this process has not been studied. Therefore, the present study aimed to explore this process. The expression levels of Notch‑1 pathway‑associated molecules were measured in response to NE using reverse transcription‑quantitative polymerase chain reaction and western blotting. Alongside NE treatment, two Notch‑1 pathway blockers, Notch‑1‑specific small interfering (si)RNA and DAPT (an inhibitor of the Notch‑1 pathway), were used to explore the relationship between NE and the Notch‑1 pathway in the development of pancreatic cell malignant biological behaviors, including cell viability, apoptosis and cell invasion. The results demonstrated that treatment with NE enhanced cell viability and invasion, and inhibited apoptosis of PDAC cells; however, these effects were suppressed following treatment with Notch‑1‑specific siRNA and DAPT. In conclusion, NE may enhance the malignant biological behaviors of PDAC via activating the Notch‑1 pathway.

Cellular antiseizure mechanisms of everolimus in pediatric tuberous sclerosis complex, cortical dysplasia, and non-mTOR-mediated etiologies.

SummaryThe present study was designed to examine the potential cellular antiseizure mechanisms of everolimus, a mechanistic target of rapamycin (mTOR) pathway blocker, in pediatric epilepsy cases. Cortical tissue samples obtained from pediatric patients (n = 11, ages 0.67–6.75 years) undergoing surgical resections for the treatment of their pharmacoresistant epilepsy were examined electrophysiologically in ex vivo slices. The cohort included mTOR‐mediated pathologies (tuberous sclerosis complex [TSC] and severe cortical dysplasia [CD]) as well as non–mTOR‐mediated pathologies (tumor and perinatal infarct). Bath application of everolimus (2 μm) had practically no effect on spontaneous inhibitory postsynaptic activity. In contrast, long‐term application of everolimus reduced spontaneous excitatory postsynaptic activity, burst discharges induced by blockade of γ‐aminobutyric acid A (GABAA) receptors, and epileptiform activity generated by 4‐aminopyridine, a K+ channel blocker. The antiseizure effects were more pronounced in TSC and CD cases, whereas in non–mTOR‐mediated pathologies, the effects were subtle at best. These results support further clinical trials of everolimus in mTOR pathway–mediated pathologies and emphasize that the effects require sustained exposure over time.

RETRACTED: DUSP1 alleviates cerebral ischaemia reperfusion injury via inactivating JNKMff pathways and repressing mitochondrial fission

Evidence continues to accrue implicating mitochondrial fission in the aetiology of cerebral ischaemia reperfusion (IR) injury. Dual-specificity phosphatase-1 (DUSP1) has been found to be associated with mitochondrial protection in several diseases. We aimed to explore the functional role of DUSP1 in cerebral IR injury, focusing on its influence on mitochondrial fission. WT mice and DUSP1 transgenic mice were subjected to cerebral IR in vivo. In vitro, the hypoxia-reoxygenation model was used with N2a cells to mimic cerebral IR injury. Mitochondrial function was monitored via western blotting and immunofluorescence. Pathway blocker was used to establish the role of the JNK-Mff signaling pathway in mitochondrial fission. DUSP1 expression is downregulated after cerebral IR injury, and overexpression of DUSP1 could significantly reduce the infarction area and attenuate neuronal death exerted by cerebral IR injury. In vitro, we found that HR injury induced neuronal mitochondrial damage via augmenting mitochondrial fission. DUSP1 overexpression inhibited mitochondrial fission, thereby preserving mitochondrial homeostasis against HR injury. Furthermore, our data illustrated that DUSP1 regulated mitochondrial fission via JNK-Mff pathways. Increased DUSP1 prevented JNK activation, upregulated Yap expression, and suppressed Mff phosphorylation, reducing the formation of mitochondrial fragmentation. However, re-activation of JNK-Mff pathways abrogated the inhibitory effect of DUSP1 overexpression on mitochondrial fission. This finding first uncovers that DUSP1 downregulation could be considered the primary reason for cerebral IR injury via evoking deleterious mitochondrial fission and activating harmful JNK-Mff pathways.

TAZ inhibition promotes IL-2-induced apoptosis of hepatocellular carcinoma cells by activating the JNK/F-actin/mitochondrial fission pathway

BackgroundCytokine-based cancer therapies have attracted a great deal of attention in recent years. Unfortunately, resistance to treatment limits the efficacy of these therapeutics. Therefore, the aim of our study was to explore the mechanism of IL-2-based therapy for hepatocellular carcinoma in an attempt to increase the efficiency of this treatment option.MethodsHepG2 cells were treated with IL-2. Then, siRNA against TZA was used to transfected into HepG2 cells. Cellular apoptosis was measured via MTT assay, TUNEL assay and caspase-3 activity. Cellular proliferation was evaluated via EdU assay and western blotting. Cellular migration was detected via Transwell assay. Mitochondrial function was monitored by mitochondrial potential analysis, ROS staining, immunofluorescence and western blotting. Pathway blocker and activator were used to establish the role of JNK/F-actin/mitochondrial fission signaling pathway in HepG2 cells stress response.ResultsOur study found that IL-2 treatment significantly reduced the viability, mobility and proliferation of HepG2 cells in vitro. We also demonstrated that IL-2 treatment was accompanied by an increase in the expression of transcriptional co-activator with PDZ-binding motif (TAZ). Interestingly, genetic ablation of TAZ in the presence of IL-2 further promoted apoptosis, inhibited mobility, and arrested proliferation in HepG2 cells. At the molecular level, IL-2 administration activated excessive mitochondrial fission via the JNK/F-actin pathway; these effects were further enhanced by TAZ deletion. Mechanistically, TAZ knockdown further increased the expression of mitochondrial fission-related proteins such as Drp1, Mff and Fis. The augmented mitochondrial fission stimulated ROS overproduction, mediated redox imbalance, interrupted mitochondrial energy generation, reduced mitochondrial membrane potential, promoted leakage of the pro-apoptotic molecule cyt-c into the nucleus, and initiated caspase-9-related mitochondrial death. Further, we demonstrated that the anti-proliferative and anti-metastatic effects of IL-2 in HepG2 cells were enhanced by TAZ deletion, suggesting that IL-2 sensitizes HepG2 cells to IL-2-based cytokine therapy. However, JNK/F-actin pathway blockade could abrogate the inhibitory effects of TAZ deletion on HepG2 migration, proliferation and survival.ConclusionsTaken together, our data indicate that the anti-tumor effects of IL-2-based therapies may be enhanced by TAZ deletion in a JNK/F-actin pathway-dependent manner. This finding provides a novel combinatorial therapeutic approach for treating hepatocellular carcinoma that might significantly increase the efficacy of cytokine-based therapies in a clinical setting.

Abstract 3632: Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models

Abstract Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3632.

Penehyclidine hydrochloride regulates mitochondrial dynamics and apoptosis through p38MAPK and JNK signal pathways and provides cardioprotection in rats with myocardial ischemia–reperfusion injury

AimThe potential mechanism of penehyclidine hydrochloride (PHC) against myocardial ischemia–reperfusion (I/R) injury has not been fully elucidated. The aim of the present study was to reveal whether mitochondrial dynamics, apoptosis, and MAPKs were involved in the cardioprotective effect of this drug on myocardial I/R injury. MethodsNinety healthy adult male Wistar rats were separately pretreated with normal saline (0.9%); PHC; and signal pathway blockers of MAPKs, Drp1, and Bcl-2. Coronary artery ligation and subsequent reperfusion were performed to induce myocardial I/R injury. Echocardiography was performed. Myocardial enzymes and oxidative stress markers were detected. Myocardial cell apoptotic rates and infarct sizes were measured. Mitochondrial function was evaluated. Expression levels of MAPKs, mitochondria regulatory proteins (Drp1, Mfn1/2), and apoptosis-related proteins (Bcl-2, Bax) were determined. ResultsPHC pretreatment improved myocardial abnormalities (dysfunction, injury, infarct size, and apoptotic rate), mitochondrial abnormalities (dysfunction and fission), and excessive oxidative stress and inhibited the activities of p38MAPK and JNK signal pathways in rats with myocardial I/R injury (P < 0.05). Additionally, p38MAPK and JNK blockers (SB239063 and SP600125, respectively) had an effect on rats same as that of PHC. Although Drp1 blocker (Mdivi-1) showed a similar cardioprotective effect (P < 0.05), it did not affect the expression of MAPKs and apoptosis-related proteins (P > 0.05). In addition, Bcl-2 blocker (ABT-737) caused a high expression of Drp1 and a low expression of Mfn1/2 (P < 0.05). ConclusionPHC regulated mitochondrial dynamics and apoptosis through p38MAPK and JNK signal pathways and provided cardioprotection in rats with myocardial I/R injury.

Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.

Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.

Roles of methyltrienolone (R1881) in AKTs and AR expression patterns of cultured granulosa-lutein cells.

AR-mediated androgen signaling plays a key role in female reproductive system. Granulosa-lutein cells (GCs) are the main sites for expression of androgen receptor (AR). There is also a close relation between AKT signaling and AR. Here, we assayed the role for a synthetic AR ligand methyltrienolone (R1881) in expressions of AKTs and AR. Controlled ovarian hyperstimulation (COH) was performed in 20 normal women. Mural GCs were isolated by filtration method, cultured, and passaged. Then, the cells were starved for 48 h with 10% charcoal stripped FBS. The cells were then treated with R1881, bicalutamide (AR blocker), LY294002 (PI3K/AKT pathway blocker), and combination of them for 48 h. Finally, GCs were evaluated for quantitative real-time PCR analysis of AKT1, AKT2, AKT3, and AR, and also Western blot assessment of total AKT and phosphorylated AKT (p-AKT) [Ser473 and Thr308]. Addition of R1881 to the GCs culture showed high expressions of AKT1, AKT2, and AKT3 (P ≤ 0.05 vs LY294002 group and bicalutamide group). Expressions of AKT1 and AKT2 were decreased in the GCs under exposure to bicalutamide or LY294002 (P ≤ 0.05 vs R1881). AKT1, AKT2, and AKT3 showed decreased rates of expressions in the LY294002 + bicalutamide group (P ≤ 0.05 vs R1881). AR, total AKT and p-AKT showed no significant differences between groups. Our findings indicate that 46 h exposure with R1881 could affect AKTs expressions in the GCs of pre-ovulatory phase, but it cannot promote AR expression and AKTs activation.

Protctive effects of Angelicin on UVB-induced HDF cells damage and its mechanism

Objective To study the effect of Angelicin on proliferation activity and anti-aging related protein expression of human HDF cells and its mechanism. Methods According to the random number table method, the cells were divided into blank group, model group, estradiol group, Angelicin group, estrogen receptor antagonist+estradiol group, estrogen receptor antagonist+Angelicin group, and P38 pathway blocker group. Different groups were given the according drugs respectively for 24 h. Except the blank group, all the groups of cells were given UVB irradiation with a dose of 150 mJ/cm2. The MTT assay was used to detect cell proliferation rate. The Western blot was used to detect the expression levels of COLⅠ, MMP-1, ERβ, P38 and p-P38 in cells, and the MMP-1 mRNA expression was detected by real-time fluorescent quantitative PCR. Results Compared with the model group, the proliferation rate of HDF cells significantly increased in Angelicin (10, 1, 0.1 and 0.01 μmol/L groups) (P<0.01); The protein expression of COLⅠ (0.326 ± 0.006 vs. 0.176 ± 0.007), ERβ (0.281 ± 0.011 vs. 0.143 ± 0.006) significantly increased (P<0.01), and the expression of MMP-1 (0.256 ± 0.006 vs. 0.395 ± 0.006) and p-P38 (0.224 ± 0.003 vs. 0.318 ± 0.005) significantly decreased (P<0.01) in Angelicin 10 μmol/L group. Compared with 10 μmol/L Angelicin group, the protein expression of estrogen receptor antagonist+Angelicin group ERβ (0.120 ± 0.007 vs. 0.281 ± 0.011) significantly decreased and MMP-1mRNA (1.377 ± 0.012 vs. 1.024 ± 0.010) significantly increased (P<0.01). Conclusions The Angelicin may degrade MMP-1 through the ER-P38 MAPK signaling pathway,and then promote collagen synthesis, to achieve the purpose of prevention and treatment of photoaging. Key words: Angelicin; Estrogen receptor beta; p38 mitogen-activated protein kinases; Collagen; Matrix metalloproteinases; Skin aging