Early Phase Of Apoptosis Research Articles

Anti‑epileptic mechanism of isopimaric acid from Platycladi cacumen based on network pharmacology, molecular docking and biological validation.

Platycladi cacumen (PC) is derived from the dry twigs and leaves of Platycladi orientalis (L.) Franco and exerts anti-epileptic effects. However, its mechanism of action remains unknown. The present study explored the potential anti-epileptic components and mechanisms of PC. The primary active components and targets of PC were analyzed using network pharmacology and a lipopolysaccharide (LPS)-induced murine microglial cell line (BV2) was used to confirm anti-epileptic effects by detecting reactive oxygen species (ROS), apoptosis, inflammatory markers, cell migration and signaling pathways. A total of 13 core active components showed druggable properties, of which deoxypicrop odophyllotoxin, hinokinin and isopimaric acid (IPA) were predicted to cross the blood-brain barrier. In total, 255 potential targets of these three compounds were predicted using SwissTargetPrediction and Similarity Ensemble Approach websites and 150 were associated with epilepsy. In vitro experiments confirmed that IPA significantly inhibited LPS-induced microglial oxidative stress and inflammation by decreasing the migration area, cellular ROS content, lactate dehydrogenase release and early phase of apoptosis. IPA also increased the mRNA expression of anti-oxidative enzymes (superoxide dismutase-1 and -2) and suppressed inflammatory cytokines (interleukin-1β and tumor necrosis factor-α). Furthermore, IPA phosphorylated AKT and mTOR proteins. Taken together, the present findings suggested that IPA is a potential anti-epileptic compound derived from PC.

β‐Cyclodextrin‐Modified Cobalt Nanoparticles as 5‐Fluorouracil Carriers

AbstractMetal nanoparticles having magnetic properties are exciting for application in the directed transport of anticancer drugs encapsulated in them. To ensure drug‐loading capacity and controlled release, surface modification with host molecules is a fascinating choice. In this paper, we report the synthesis and surface modification of magnetic cobalt metal nanoparticles with oligosaccharide host molecules (β‐cyclodextrin) tethered on the surface. A β‐cyclodextrin‐folate derivative is synthesized and characterized using Infrared‐, Nuclear Magnetic Resonance‐, and Matrix‐Assisted Laser Deposition Ionization‐Time of Flight mass spectrometry. The size of the cobalt nanoparticles carrying the host molecule on the surface is ~10 nm. The nanoparticles are characterized using X‐Ray Diffraction, Transmission Electron Microscopy, particle size analyzer, Energy‐Dispersive X‐ray, and X‐ray Photoelectron Spectroscopy. The bare magnetic nanoparticles also show a strong saturation magnetization of 142.21 emu g−1. The chemotherapeutic drug 5‐fluorouracil is loaded on the surface‐modified cobalt nanoparticles through host: guest complexation mode and its controlled release is measured in‐vitro. The slow release is spread over more than 14 days, and a complete release of the loaded therapeutic cargo is not reached. The release is pH‐dependent and changes with acidic levels of the medium. The anticancer activity of the drug‐loaded nanocarrier on MDA‐MB‐231 (breast cancer) cells is investigated. The mechanism of cell death is studied, and the cells are found inhibited in the synthesis phase. Flow cytometric analysis shows that 79.70 % of the empty nanocarrier‐treated and 74.86 % 5‐fluorouracil‐loaded nanocarrier‐treated MDA‐MB‐231 cells were in the early apoptosis phase. This new combination nanocarrier is intriguing owing to its structural and magnetic properties, and the action on cell lines.

The Effect of Radiotherapy on Cell Survival and Inflammatory Cytokine and Chemokine Secretion in a Co-Culture Model of Head and Neck Squamous Cell Carcinoma and Normal Cells.

Radiotherapy (RT) is one of the main treatments for head and neck squamous cell carcinomas (HNSCCs). Unfortunately, radioresistance is observed in many cases of HNSCCs. The effectiveness of RT depends on both the direct effect inducing cell death and the indirect effect of changing the tumor microenvironment (TME). Knowledge of interactions between TME components after RT may help to design a new combined treatment with RT. In the study, we investigated the effect of RT on cell survival and cell secretion in a co-culture model of HNSCCs in vitro. We examined changes in cell proliferation, colony formation, cell cycle phases, type of cell death, cell migration and secretion after irradiation. The obtained results suggest that the presence of fibroblasts and endothelial cells in co-culture with HNSCCs inhibits the function of cell cycle checkpoints G1/S and G2/M and allows cells to enter the next phase of the cell cycle. We showed an anti-apoptotic effect in co-culture of HNSCCs with fibroblasts or endothelial cells in relation to the execution phase of apoptosis, although we initially observed increased activation of the early phase of apoptosis in the co-cultures after irradiation. We hypothesize that the anti-apoptotic effect depends on increased secretion of IL-6 and MCP-1.

Not all benzimidazole derivatives are microtubule destabilizing agents

In recent years, microtubule-targeting agents (MTAs) have gained considerable interest in developing novel small-molecule anticancer drugs. MTAs demonstrate anticancer activity either as microtubule-stabilizing agents (paclitaxel) or microtubule-destabilizing agents (nocodazole). FDA-approved drugs containing a benzimidazole ring (nocodazole, albendazole, mebendazole, etc.) are well-known microtubule-destabilizing agents. Thus, most recent research on benzimidazole scaffold-based MTAs focuses on developing microtubule-destabilizing agents. However, there is no report on the benzimidazole scaffold-based microtubule-stabilizing agent. Here, we present the benzimidazole derivatives NI-11 and NI-18 that showed a profound anticancer activity as microtubule-stabilization agents. About twenty benzimidazole analogues were synthesized with excellent yield (80.0% ∼ 98.0%) and tested for their anticancer activity using two cancer cell lines (A549, MCF-7) and one normal cell line (MRC-5). NI-11 showed IC50 values of 2.90, 7.17, and 16.9 µM in A549, MCF-7, and MRC-5 cell lines. NI-18 showed IC50 values of 2.33, 6.10, and 12.1 µM in A549, MCF-7, and MRC-5 cell lines. Thus, NI-11 and NI-18 demonstrated selectivity indexes of 5.81 and 5.20, respectively, which are much higher than the currently available anticancer agents. NI-11 and NI-18 inhibited the cancer cell motility and migration, induced the early phase apoptosis. Both of these comounds were found to show an upregulation of DeY-α-tubulin and downregulation of Ac-α-tubulin expressions in cancer cells. Eventhough the reported benzimidazole scaffold-based commercially available drugs are known to be microtubule-destabilizing agents, the analogues NI-11 and NI-18 were found to have microtubule-stabilizing activity. The in vitro tubulin polymerization assay and the immunofluorescence assay results indicate that the NI-11 and NI-18 exhibit anticancer activity by stabilizing the microtubule network.

Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells

Abstract Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

In Vivo and in vitro antitumor activity of tomatine in hepatocellular carcinoma

Background: There is abundant ethnopharmacological evidence the uses of regarding Solanum species as antitumor and anticancer agents. Glycoalkaloids are among the molecules with antiproliferative activity reported in these species. Purpose: To evaluate the anticancer effect of the Solanum glycoalkaloid tomatine in hepatocellular carcinoma (HCC) in vitro (HepG2 cells) and in vivo models. Methods: The resazurin reduction assay was performed to detect the effect of tomatine on cell viability in human HepG2 cell lines. Programmed cell death was investigated by means of cellular apoptosis assays using Annexin V. The expression of cancer related proteins was detected by Western blotting (WB). Reactive oxygen species (ROS) and calcium were determined by 2,7-dichlorodihydrofluorescein diacetate and Fluo-4, respectively. Intrahepatic HepG2 xenograft mouse model was used to elucidate the effect of tomatine on tumor growth in vivo. Results and Discussion: Tomatine reduced HepG2 cell viability and induced the early apoptosis phase of cell death, consistently with caspase-3, -7, Bcl-2 family, and P53 proteins activation. Furthermore, tomatine increased intracellular ROS and cytosolic Ca+2 levels. Moreover, the NSG mouse xenograft model showed that treating mice with tomatine inhibited HepG2 tumor growth. Conclusion: Tomatine inhibits in vitro and in vivo HCC tumorigenesis in part via modulation of p53, Ca+2, and ROS signalling. Thus, the results suggest the potential cancer therapeutic use of tomatine in HCC patients.

Up-and-coming anti-epileptic effect of aloesone in Aloe vera: Evidenced by integrating network pharmacological analysis, in vitro, and in vivo models.

Background: Aloe vera is a medically valuable plant with anti-epileptic activity; however, its mechanism of action remains unknown. In this study, network pharmacological, in vitro, and in vivo experiments were carried out to explore the potential anti-epileptic components and targets of Aloe vera. Methods: The main active components of Aloe vera were identified by searching the Traditional Chinese Medicine System Pharmacology database. Targets of Aloe vera were predicted using SwissTargetPrediction, whereas information about the epilepsy disease targets was obtained from Gene Cards. The protein–protein interaction network and core targets were screened according to the topological structure and CytoNCA plugin. The glutamate-induced HT22 cell line and pentylenetetrazol-induced seizure rats were used to confirm the effect of aloesone by detecting reactive oxygen species (ROS) and apoptosis, and predicting the targets. Results: A total of 14 core active components were selected based on the screening criteria of oral bioavailability ≥30% and drug-likeness ≥ 0.10. Four compounds, namely linoleic acid, aloesone, isoeleutherol glucosiden qt, and anthranol, demonstrated the potential ability of crossing the blood-brain barrier. A total of 153 targets associated with epilepsy were predicted for the four compounds. Moreover, after network analysis with CytoNCA, 10 targets, namely, MAPK1, SRC, MARK3, EGFR, ESR1, PTGS2, PTPN11, JAK2, PPKCA, and FYN, were selected as the core genes, and SRC, which has been predicted to be the target of aloesone and anthranol, exhibited the highest subgraph centrality value. In vitro experiments confirmed that aloesone treatment significantly inhibited the glutamate-induced neuronal injury by reducing the intracellular ROS content and the early phase of apoptosis. Additionally, treatment with 50 mg/kg aloesone resulted in anti-seizure effects by reducing the seizure score and prolonging the latent period in acute and chronic rats. Furthermore, aloesone treatment increased the phosphorylation of c-SRC at Y418 and reduced the phosphorylation at Y529, simultaneously activating c-SRC. Conclusion: Integrating network pharmacology with in vitro and in vivo experiments demonstrated that aloesone, which inhibited seizure by activating c-SRC, is a potential anti-seizure compound present in Aloe vera.

Antioxidant and Neuroprotective Properties of Non-Centrifugal Cane Sugar and Other Sugarcane Derivatives in an In Vitro Induced Parkinson's Model.

Non-centrifugal cane sugar (NCS) is a traditional sweetener in most sugarcane regions of the world. In Colombia, this product has a socio-economic importance due to the extensive cultivation area and the high consumption rate per capita. NCS traditional processing involves consecutive stages of thermal processing that begin with juice extraction, clarification, evaporation, and finish with syrup crystallization into a solid commercial product, identified as NCS. Sugarcane is known to have a natural content of polyphenols, amino acids, vitamins, minerals, and complex sugars, some of which are reported as antioxidant and antiproliferative agents thought to be responsible for the product’s bioactive profile. There is evidence to suggest that traditional thermal processing to obtain NCS leads to a considerable decrease in the contents of these bioactive compounds, mainly due to uncontrolled process variables such as temperature. Accordingly, the aim of this study was to assess and compare the bioactivity of sugarcane (SC) derivatives produced under controlled thermal conditions versus the traditional method. To achieve this goal, we evaluated the cytotoxic, antioxidant, and neuroprotective effects of varying concentrations of SC derivatives in an in vitro induced Parkinson’s model. Results demonstrate non-cytotoxic activity on the cellular model by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LDH assays, even at the highest tested concentration of 8 mg/mL, for all SC derivatives. The effect of SC derivatives on the induced oxidative stress model showed a biological reversion and recovering effect of the mitochondrial membrane potential and a halting of the progress into the early apoptosis phase. In conclusion, we demonstrated that the bioactive compounds present in SC derivatives obtained by a process under controlled temperature conditions are largely preserved, and even their biological activities are enhanced compared with SC derivatives obtained by the traditional thermal evaporation of SC-juice.

Imaging flow cytometry and fluorescence microscopy in assessing radiation response in lymphocytes from umbilical cord blood and cancer patients.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Abstract 6263: Hypoxia-targeting prodrug approach for multimodal chemotherapy and immunogenic cell death in aggressive triple negative breast cancer

Abstract Immune checkpoint blockade molecules, including antibodies against PD-1 and PD-L1 have gained much clinical recognition in the past few years. These strategies include harnessing the host's immune system to invade cancer cells by inhibiting the interaction between PD-1 and its ligand, PD-L1, thereby facilitating the activation of CD8+ T cells to mount an attack on cancer cells. Triple Negative Breast Cancer (TNBC) accounts for 10-20% of the total breast carcinoma cases. There is no FDA-approved targeted therapy for TNBC due to lack of the major biomarkers: estrogen, progesterone and HER-2. TNBC is difficult to treat, with a high rate of metastasis to nearby organs. We have identified the role of a hypoxia biomarker, carbonic anhydrase IX (CAIX) in proliferation and metastasis of TNBC. Based on this observation, we developed a CAIX-targeted Doxorubicin (Dox) prodrug nanoparticle, abbreviated as CAIX-Dox-NP. CAIX-Dox-NP can deliver the chemotherapeutic, Dox payload selectively in hypoxic tumor microenvironment, thus reducing Dox-associated cardiotoxicity. Most of the advanced stages TNBC solid tumors present themselves with oxygen-deficient, vascularized and matrix-rich core which is impermeable to most of the chemotherapy drugs. CAIX-Dox-NP has the ability to penetrate deep into the TNBC and kill epithelial tumor cells as well as tumor-growth promoting T-cells. CAIX-Dox-NP showed greater penetration in the core of hypoxic TNBC spheroid. Recent studies showed that Immunogenic Cell Death (ICD) has vital role in resurrecting the CD8+ T-cells mediated tumor killing. Combination of anti-PD-1 with inducers of ICD like Dox accentuate the ability of CD8+ T cells in killing of cancer cells. Our approach is to utilize CAIX-Dox for achieving CAIX-mediated targeted delivery and multimodal chemotherapy in addition to induction of ICD. Preliminary studies with CAIX-Dox has profound effect of killing TNBC cells, 4T1, through induction of early phase apoptosis. The Western Blot analysis in 4T1 treated with CAIX-Dox has proved the significant upregulation of ICD biomarkers, HMGB1 and calreticulin, by greater than 3-fold compared to control. To improve the bioavailability of CAIX-Dox, we developed CAIX-Dox-NP that can hitchhike on the circulating plasma albumin. The nanoparticles travel to the tumor and deliver the CAIX-Dox at the hypoxic core of TNBC, resulting in induction of apoptosis and ICD. Inspired from this outcome, we are developing a model for coculture of cancer and T-cells for evaluating if CAIX-Dox treatment can reeducate T-cells in killing cancer cells through ICD pathway. Citation Format: Ketki Bhise, Samaresh Sau, Mohd Ahmar Rauf, Arun K. Rishi, Arun K. Iyer. Hypoxia-targeting prodrug approach for multimodal chemotherapy and immunogenic cell death in aggressive triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6263.

Coordinated changes in cell membrane and cytoplasm during maturation of apoptotic bleb.

Apoptotic cells form membrane blebs, but little is known about how the formation and dynamics of membrane blebs are regulated. The size of blebs gradually increases during the progression of apoptosis, eventually forming large extracellular vesicles called apoptotic bodies that have immune-modulating activities. In this study, we investigated the molecular mechanism involved in the differentiation of blebs into apoptotic blebs by comparing the dynamics of the bleb formed during cell migration and the bleb formed during apoptosis. We revealed that the enhanced activity of ROCK1 is required for the formation of small blebs in the early phase of apoptosis, which leads to the physical disruption of nuclear membrane and the degradation of Lamin A. In the late phase of apoptosis, the loss of asymmetry in phospholipids distribution caused the enlargement of blebs, which enabled translocation of damage-associated molecular patterns to the bleb cytoplasm and maturation of functional apoptotic blebs. Thus, changes in cell membrane dynamics are closely linked to cytoplasmic changes during apoptotic bleb formation.

Hypoxia Targeting Nanoparticles for Immunogenic Cell Death of Triple Negative Breast Cancer

Triple Negative Breast Cancer (TNBC) accounts for 10–20% mortality amongs all breast carcinoma cases. There is no targeted therapy approved for TNBC owing to absence of major biomarkers: estrogen, progesterone and Her‐2. Advanced stage TNBC tumors present themselves with a hypoxic, vascularized drug‐resistant core, which is rich in cancer stem cells and tumor‐associated growth factors. Earlier reports from our lab have identified the role of hypoxia biomarker, carbonic anhydrase IX (CAIX) in tumor proliferation and metastasis. Based on this, we have developed a CAIX‐targeted prodrug of Doxorubicin (CAIX‐Dox). Further, we have encapsulated the prodrug in long circulating, serum albumin binding lipid nanoparticles (abbreviated CAIX‐Dox‐NP) that can selectively deliver Dox to the hypoxic regions of the tumor, thus reducing Dox‐associated toxicity. CAIX‐Dox‐NP were synthesized using high pressure homogenization for achieving nanosized particles with a narrow polydispersity index. Nanoparticles with size ~200 nm and CAIX‐Dox loading of 13–19% were obtained, which were stable over 48 h at 10% serum and for over 3 months at refrigerated conditions. CAIX‐Dox‐NP were optimized based on lipid composition and ratio of aqueous to organic phases. CAIX‐Dox exhibited a sustained release pattern at physiological and mildly acidic conditions in vitro. Recent studies have shown that immunogenic cell death (ICD) plays an important role in resurrecting CD8+ T cells mediated tumor killing. ICD inducers like Dox, when presented as hypoxia targeting prodrug in the form of CAIX‐Dox, has the ability to penetrate deep into the tumor tissues, including hypoxic regions, killing epithelial cells as well as tumor promoting T cells. CAIX‐Dox has shown killing of syngeneic 4T1 TNBC cells via ICD pathway as demonstrated by Western blot analysis through upregulation of ICD markers like HMGB1, calreticulin and induction of early phase apoptosis on treatment over 72 h, by a factor greater than 3‐fold as compared to control. Thus far, CAIX‐Dox portend to be a new avenue for the treating TNBC, warranting further investigations.

Transcriptome dynamics of alternative splicing events revealed early phase of apoptosis induced by methylparaben in H1299 human lung carcinoma cells.

Methylparaben is most frequently used as an antimicrobial preservative in pharmaceuticals and foods. Methylparaben has been subjected to toxicological studies owing to the increasing concern regarding its possible impact on the environment and human health. However, the cytotoxicity and underlying mechanisms of methylparaben exposure in human lung cells have not been explored. Here, we investigated the effect of methylparaben on cell cycle, apoptotic pathways, and changes in the transcriptome profiles in human lung cells. Our results demonstrate that treatment with methylparaben causes inhibition of cell growth. In addition, methylparaben induced S- and G2/M-phase arrest as a result of enhanced apoptosis. Transcriptome analysis using RNA-seq revealed that mRNA expression of ER stress- and protein misfolding-related gene sets was upregulated in methylparaben-treated group. RNA splicing- and maturation-related gene sets were significantly down-regulated by methylparaben treatment. Interestingly, RNA-seq analysis at the transcript level revealed that alternative splicing events, especially retained intron, were markedly changed by a low dose of methylparaben treatment. Altogether, these data show that methylparaben induces an early phase of apoptosis through cell cycle arrest and downregulation of mRNA maturation.

Inhibitory Effect and Molecular Mechanism of the New Phorphyrin-Based HCE6 Photosensitizer on the Activity of MKN45 Human Gastric Cancer Cells.

Gastric cancer is the fourth most common cancer worldwide and the third most common in Asia, with a high mortality. Photodynamic therapy (PDT) is a new treatment for cancer. With advantages of minimum invasiveness, small adverse side effects and high selectivity, PDT can be used as palliative treatment for patients with advanced gastric cancer. YLG I, also known as 2-(1 hexyloxyethyl)-2-devinyl porphin e6 trisodium salt (HCE6), is a recently developed photosensitizer. A previous study showed that HCE6 significantly inhibited the growth of QBC939 human cholangiocarcinoma cells. However, the effects and mechanisms of HCE6 on gastric cancer cell suppression are not known. In this study, we investigated the effects of HCE6 on the human gastric cancer cell line MKN45 and found that at the concentration of 2.0 mg/L, HCE6 almost completely killed MKN45 cells at a light intensity of 3.6 J/cm². RNAseq results confirmed that mitochondria and endoplasmic reticulum (ER)-mediated apoptosis was involved in the effects of HCE6 on cell death, and we also found that HCE6 induced chromosome conformational changes in the early phase of apoptosis. The results of our study help elucidate the molecular mechanisms underlying HCE6-mediated inhibition of gastric cancer cell growth and provide a theoretical basis and molecular targets for the treatment of gastric cancer.

Anticancer Activity of Polyisoprenoids from Avicennia alba Blume. in WiDr Cells.

Colorectal cancer is the third most common cancer world wide and has been occurred more in developing regions. The use of conventional chemotherapy agents may lead to various adverse effects. Therefore, it is required to find the potential drug for anticancer from alternative source of natural product including mangrove plants. The present study was conducted to determine the anticancer activity of polyisoprenoids from Avicennia alba Blume. leaves (PAL) in WiDr cells. Cell cycle inhibition, apoptosis activity, and suppression of cyclooxygenase-2 (COX-2) were also evaluated. The anticancer activity of PAL was determined by observing the activity of these compounds against WiDr cells using the [3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromida] MTT assay. Inhibition of the cell cycle and increased apoptosis were analysed by flowcytometry. Suppression of COX-2 was analysed using immunocytochemistry. PAL exhibited anticancer activity against WiDr cells with an IC50 of 173.775 μg/mL. Cell cycle analysis revealed that the inhibition occurred in the G0-G1 phase, and apoptosis occurred in the early apoptosis phase. Furthermore, the result of an analysis of COX-2 expression showed that PAL enabled the suppression of COX-2 expression. PAL can be used as anticancer agents against WiDr colon cancer cells. However, in-vivo studies is required to confirm the in-vitro finding of the anticancer activity of polyisoprenoid extract.

Detection of the Calcium and ATP Role in Apoptosis of Retinoblastoma Culture Cells through Caspase-3 Expression

Retinoblastoma is a malignant retinal tumor associated with apoptotic deregulation. Retinoblastoma cells are sensitive to NK cells. These cells can stimulate apoptosis. Apoptosis in retinoblastoma occurs in the early phase and is paradoxical. The increasing amounts of Bcl-2 and Caspase-3 as apoptotic executors are inversely proportional to apoptosis. Calcium and ATP as second messengers and signaling molecules play role in mediating cell responses including retinoblastoma cell development and death. Apoptosis requires sufficient energy from ATP and its mediated by Calcium. This study aimed to perceive the effects of Calcium and ATP in the process of retinoblastoma cell death through Caspase-3 pathway. The subjects were poorly differentiated retinoblastoma cell cultures treated with NK cells (treatment group) compared to those which were not exposed to NK cells (control group). Through the cell flowcytometry test that expresses Caspase-3 and apoptosis is calculated, meanwhile the levels of Calcium and ATP activity produced during the apoptosis process are quantitatively calculated. Examination of ATP activity uses a colorimetric method while the calcium content is calculated using a clinical chemistry system. Calcium and ATP were negatively correlated at 27.4% (p <0.05). ATP also showed a very significant negative correlation of 75.8% (p <0.01) against Caspase-3 and is significantly positively correlated with apoptosis of 46.8% (p,0.05). Whereas Caspase-3 is negatively correlated with apoptosis by 46.6% (p <0.05). In the Caspase-3 pathway, allogeneic NK cell administration in retinoblastoma cells increases the level of Calcium which plays a role in the early phase apoptosis process, whereas ATP which was formed is insufficient to cause maximal apoptosis due to extracellular calcium entry into NK cells. Extracellular ATP does not play a role in the induction of apoptosis in retinoblastoma cells treated with NK cells.

Adhesion of Thymocytes to the Thymic Epithelial Cells and Participation of Neyropilin-1 and Plexin A1 in the Adhesion

Thymic epithelial cells play not only an important structural role, but also create the microenvironment necessary for the maturation and differentiation of thymocytes. One of the stages of maturation of thymocytes is their adhesion to the epithelial cells of the thymus, the mechanism of which has not been sufficiently studied. The goal of the study was investigation of the interaction of mouse thymocytes with cortical cTEC1-2 and the medullary mTEC3-10 thymic epithelial cell lines, namely, the dynamics of the adhesion, apoptosis, and population composition, as well as the expression of the neuropilin-1 (Nrp-1) and plexin A1 receptors (PlexA1) among adherent, nonadherent, and control cells cultured without epithelium. The maximal adhesion index was observed for a 30-min cocultivation; after this time, the index decreased. The main population of adherent cells consisted from immature CD4+CD8+ lymphocytes, and the relative content of mature CD4+ and CD8+ thymocytes was lower than in the control. We have shown for the first time that, even at short cocultivation periods (30 and 120 min), an increase in the content of thymocytes at the early apoptosis phase was observed among the population of adherent cells. For the first time, data have been obtained demonstrating the participation of Nrp-1 and PlexA1 in the process of thymocyte adhesion to mouse thymic epithelial cells as independent molecules in the absence of their semaphorin 3A ligand. It was shown that the expression of Nrp-1 on the surface of adherent thymocytes was significantly reduced during the adhesion process compared to the control, and the expression of PlexA1 increased. Preincubation of thymocytes with antibodies to Nrp-1 increased their adhesion to epithelial cells. The data obtained contribute to a better understanding of intercellular relationships in the thymus gland.

Multiplexed imaging detection of live cell intracellular changes in early apoptosis with aggregation-induced emission fluorogens

Apoptosis is an important process for maintaining tissue homeostasis and eliminating abnormal cells in multicellular organisms. Abnormality in apoptosis often leads to severe diseases such as cancers. Better understanding of its mechanisms and processes is therefore important. Accompanying molecular biology events of apoptosis is a series of cellular morphology changes: nucleus condensation, cell shrinkage and rounding, cell surface blebbing, dynamic blebbing, apoptotic membrane protrusions and nucleus fragmentations and finally, the formation and release of apoptotic bodies. It is difficult to detect cellular changes in the early phase of apoptosis due to the subtle changes at this phase. In the current study, we induced apoptosis in HeLa cells with H2O2 and used nuclear dye Hoechst 33258, mitochondria, lysosome and cytoplasmic protein specific aggregation-induced emission fluorogens (AIEgens), TPE-Ph-In, 2M-DABS and BSPOTPE to successfully perform live cell multiplexed imaging to investigate early apoptosis cellular events. We showed the gradual dissipation of mitochondria membrane potential until it is non-detectable by TPE-Ph-In. Increased mitophagy detected by TPE-Ph-In and 2M-DABS, condensed nucleus detected by Hoechst 33258, increased permeability and/or reduced integrity of nuclear membrane, and increased intracellular vesicles detected by 2M-DABS are some of the early events of apoptosis.

Differential Effects of Quercetin and Two of Its Derivatives, Isorhamnetin and Isorhamnetin-3-glucuronide, in Inhibiting the Proliferation of Human Breast-Cancer MCF-7 Cells.

Quercetin (Que) has consistently been reported to be useful cytotoxic compound in vivo and in vitro, but little is known on its metabolites. Here, we examined and compared the cytotoxic effects of Que and its water-soluble metabolites, isorhamnetin (IS) and isorhamnetin-3-glucuronide (I3G), in human breast-cancer MCF-7 cells to uncover their tumor-inhibitory mechanisms and structure-function relationships. The results showed that Que, IS, and I3G could dose-dependently inhibit the growth of MCF-7 cells, and the cytotoxic effect was ranked as Que > IS > I3G. Furthermore, Que, IS, and I3G mediated cell-cycle arrest principally in S phase, followed by a decrease in the number of cells in G0/G1 and G2/M; moreover, 70.8, 68.9, and 49.8% of MCF-7 tumor cells entered early-phase apoptosis when treated with 100 μM Que, IS, and I3G for 48 h, respectively. Moreover, induction of apoptosis by Que, IS, and I3G was accompanied by the marginal generation of intracellular reactive oxygen species (ROS). Given these results, Que, IS, and I3G possess strong cytotoxic effects through an ROS-dependent apoptosis pathway in MCF-7 cells.

Hyaluronidase Enzyme-responsive Targeted Nanoparticles for Effective Delivery of 5-Fluorouracil in Colon Cancer.

In this study, we have successfully prepared the hyaluronic acid (HA)-conjugated mesoporous silica nanoparticles loaded with 5-fluorouracil (5-FU) to increase the anticancer efficacy in colon cancers. The particles were nanosized and perfectly spherical. In vitro release kinetics clearly showed the enzyme-sensitive release of 5-FU from HA-conjugated 5-FU loaded mesoporous silica nanoparticles (HA/FMSN). The presence of HA on the surface of nanoparticles targeted the CD44 receptors overexpressed in the colon cancer cells In vitro cell viability and apoptosis assay clearly showed the superior anticancer effect of HA/FMSN in HT29 colon cancer cells. HA/FMSN exhibited a remarkably higher 43% of cells in early apoptosis phase and 55% of cells in late apoptosis phase indicating the superior anticancer effect of HA/FMSN. HA/FMSN exhibited a significant reduction in the tumor burden compared to that of any group. HA/FMSN was 3-fold more effective than free drug and 2-fold more effective than -FU loaded mesoporous silica nanoparticles (FMSN). Overall, results suggest that the novel delivery strategy could hold enormous potential in colon cancer targeting.