Hypoxia and Proton microbeam: Role of Gap Junction Intercellular Communication in Inducing Bystander Responses on Human Lung Cancer Cells and Normal Cells

Radiation-induced bystander effect (RIBE) has been identified as an important contributing factor to tumor resistance and normal tissue damage. However, the RIBE in cancer and normal cells under hypoxia remain unclear. In this study, confluent A549 cancer and WI-38 normal cells were subjected to condition of hypoxia or normoxia, before exposure to high-LET protons microbeam. After 6 h incubation, cells were harvested and assayed for colony formation, micronucleus formation, chromosome aberration and western blotting. Our results show that there were differences of RIBE in bystander A549 and WI-38 cells under hypoxia and normoxia. The differences were also observed in the roles of HIF-1α expression in bystander A549 and WI-38 cells under both conditions. Furthermore, inhibition of gap junction intercellular communication (GJIC) showed a decrease in toxicity of hypoxia-treated bystander A549 cells, but increased in bystander WI-38 cells. These findings clearly support that GJIC protection of bystander normal cells from toxicity while enhancing in bystander cancer cells. Together, the data show a promising strategy for high-LET radiation in designing an entire new line of drugs, either increase or restore GJIC in bystander cancer cells which in turn leads to enhancement of radiation accuracy for treatment of hypoxic tumors.

Connexin Expression Is Altered in Liver Development of Yotari (dab1 -/-) Mice

Disabled-1 (Dab1) protein is an intracellular adaptor of reelin signaling required for prenatal neuronal migration, as well as postnatal neurotransmission, memory formation and synaptic plasticity. Yotari, an autosomal recessive mutant of the mouse Dab1 gene is recognizable by its premature death, unstable gait and tremor. Previous findings are mostly based on neuronal abnormalities caused by Dab1 deficiency, but the role of the reelin signaling pathway in nonneuronal tissues and organs has not been studied until recently. Hepatocytes, the most abundant cells in the liver, communicate via gap junctions (GJ) are composed of connexins. Cell communication disruption in yotari mice was examined by analyzing the expression of connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 during liver development at 13.5 and 15.5 gestation days (E13.5 and E15.5). Analyses were performed using immunohistochemistry and fluorescent microscopy, followed by quantification of area percentage covered by positive signal. Data are expressed as a mean±SD and analyzed by one-way ANOVA. All Cxs examined displayed a significant decrease in yotari compared to wild type (wt) individuals at E13.5. Looking at E15.5 we have similar results with exception of Cx37 showing negligible expression in wt. Channels formation triggered by pathological stimuli, as well as propensity to apoptosis, was studied by measuring the expression of Pannexin1 (Panx1) and Apoptosis-inducing factor (AIF) through developmental stages mentioned above. An increase in Panx1 expression of E15.5 yotari mice, as well as a strong jump of AIF in both phases suggesting that yotari mice are more prone to apoptosis. Our results emphasize the importance of gap junction intercellular communication (GJIC) during liver development and their possible involvement in liver pathology and diagnostics where they can serve as potential biomarkers and drug targets.

Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer

Breast cancer (BC) is a global public health burden, constituting the highest cancer incidence in women worldwide. Connexins 43 proteins propagate intercellular communication, gap junction intercellular communication (GJIC), remarkably expressed in several tumor types including liver, prostate, and breast. This domain of Cx43 possesses functionally critical sites identical to those involved in gating of channel and phosphorylation sites for various kinases. However, the mechanism by which Cx43 down regulation occurs in breast cancer is far from clear. Several mechanisms like Cx43 promoter hyper-methylation or a cancer-specific reduction of Cx43 expression/trafficking by the modulation of various components of the Cx43 life cycle give the idea to be involved in the down regulation of Connexins in mammary glands, but irreversible mutational alterations have not yet been proved to be among them. Summarily, the efficacy or specificity of these drugs can be increased by a combinatory approach considering an effect on both the Connexins and their regulatory molecules. This chapter will summarize the knowledge about the connexins and gap junction activities in breast cancer highlighting the differential expression and functional dynamics of connexins in the pathogenesis of the disease.

Electroacupuncture Alleviates Functional Dyspepsia by Targeting miR-221-3p to Regulate c-kit/SCF and Raf/Erk Signaling

Background: Functional dyspepsia (FD) is a gastrointestinal disorder associated with epigastric pain and fullness, with symptoms such as anxiety and depression. Electroacupuncture (EA) has emerged as a potential therapeutic strategy to treat FD via electrical stimulation at specific acupoints of the body and has previously been demonstrated to be effective in promoting gastric motility and attenuating the symptoms of FD. However, the mechanisms underlying the effect of EA remain elusive.Results: Herein, we constructed an in vivo model of FD in rats and performed EA on FD-induced rats. Preliminary bioinformatic analysis through RNA sequencing revealed that differentially expressed microRNAs exist between non-treated and EA-treated rats subjected to EA. Among them, miR-221-3p was associated with the target gene c-kit, the expression of which is closely linked to interstitial cells of Cajal (ICCs) and gap junction intercellular communication, factors critically implicated in gastrointestinal motility. We further proved that miR-221-3p overexpression aggravated FD, but EA downregulated miR-221-3p to alleviate the symptoms of FD. The mechanisms of action underlying the effect of EA involve an increase in c-kit+ ICCs, upregulation of stem cell factor and connexin 43, and enhanced Raf/Erk signaling.Conclusions: Our findings suggest that EA is an effective method of managing FD by regulating miR-221-3p, the c-kit/stem cell factor axis, and Raf/Erk signaling, reinforcing the strong role of gap junction intercellular communication in gastrointestinal motility.

Substrate ligand density modulates gap junction intercellular communication during mesenchymal cell condensation

Gap junction intercellular communication (GJIC) provides a continuous and efficient flow of biological information during tissue formation and is essential to sustain homeostasis and function in living organisms. Cell-matrix interactions have been widely addressed and their influence on tissue organization is recognized. However, how extracellular matrix (ECM) adhesion affects intercellular communication during tissue formation remains elusive. Here we use substrates with uneven nanopatterns of adhesive ligand arginine-glycine-aspartic acid (RGD) to control cell adhesion during mesenchymal condensation, a prevalent morphogenetic transition. We show that the establishment of GJIC is an adhesion-gated mechanism, which is dynamically regulated. Substrate effects continuously propagate into the forming tissue through actomyosin contraction, affecting both the 3D architecture and functionality of the GJIC network developing in prechondrogenic condensates.