The GJ-dependent as well as -independent antitumor activity of the Cx43 gene has been extensively investigated in relation to the ART drug. Flow cytometry analysis of the cytotoxic effect of ART in MCF7 and Cx43-MCF7 cells determined by PE Annexin V and 7-AAD staining.
Introduction and Literature Review 1
Introduction 3
The cancer cell for the most part resembles unicellular organisms in their functionality and regulation, which survive by uncontrolled cell proliferation and cannot terminally differentiate. Cancer cells, which usually have abnormal apoptosis responses, do not differentiate terminally, do not have growth control, and do not inhibit contact, often do not appear to do so.
Cell-Cell Communication in Carcinogenesis 4
Cells can have defective GJIC in several cases as follows, a) they may lack functional GJ, b) they may have functional GJs among themselves but are unable to communicate between non-transformed cells, c) they may lack functional cell- form cell junctions but are unable to respond to the GJ signals that mediate differentiation, opening and closing of GJ and phenotype of the cell.
The Connexin Multigene Family 6
The second system classified connexins into subclasses (a, h, g, or y) based on their sequence similarity and length of the cytoplasmic domain with added prefix “GJ” for GJ and assigned a number based on their order of discovery. Of all the connexins (Table 1.1), most established cell lines express Cx43, while an easily detectable amount of other connexins are found in very few cell lines.
Loss of GJIC and Connexins is an Early Event of Malignancy 9
Apart from the basic regulatory mechanism, such as post-translational modifications, translational control, mRNA splicing and transcription, which apply to most of the proteins, Cxs functions are also regulated at different levels of control. Furthermore, GJIC can be affected by opening and closing of the channels under different circumstances, growth factors, extracellular matrix as well as cell adhesion molecules and mutations in Cxs genes.
Tumour Suppression by Connexins 10
The mechanistic aspects of the GJ-independent regulation of cancer cells by connexin proteins depend on its role in influencing the tumorigenic cell cycle and signaling. In other reports, the overexpression of Cx43 has been correlated with the accumulation of the hypophosphated retinoblastoma (Rb) protein and an increase in the level of p27 proteins, a cyclin-dependent kinase inhibitor [52].
Restoring Connexin Expression as a Therapeutic Approach 13
Histone deacetylase (HDAC), a group of enzymes that effect global gene expression and regulate chromatin remodeling, are the targets of anti-cancer therapeutics. In prostate cancer cells, the Cx43 expression and GJIC were restored using another HDACi, trichostatin A (TSA) [ 68 ].
Drug Delivery 16
Similarly, the impact of various GJ inhibitors (GJI) such as α-glycyrrhetinic acid, or activators (GJA) such as the quinoline derivative PQ1 is still poorly defined.
Key Areas and Scope of Research 17
The present work 18
Improvement in the cytotoxic activity of the F186W mutant relative to Cx43 has been reported. The fabrication of a versatile 4-PB-linked nanoparticle composite has been demonstrated and the efficacy of the 4-PB-linked nanocarrier has also been evaluated in the spheroid-based cancer cell model.
Zaidan-Dagli, Defective intercellular communication between gap junctions in the carcinogenic process, Biochimica et biophysica acta. Kang, Effects of the histone deacetylase inhibitors sodium butyrate and trichostatin A on the inhibition of gap junctional intercellular communication by H2O2 and 12-O-tetradecanoylphorbol-13-acetate in rat liver epithelial cells, Cancer Lett.
Connexin-43 enhances tumour suppressing activity of arte-
Introduction 27
Due to the adverse side effects of the chemotherapeutic drugs, plant-based active anticancer compounds may offer a better alternative. The GJIC that developed between the neighboring cells has also been studied and assisted in bystander killing of the cells after ART treatment.
Materials and Methods 28
The proliferation of MCF7 and CX43-MCF7 cells was measured by flow cytometric analysis of CFSE (carboxyfluorescein diacetate, succinimidyl ester or CFDA-SE) loaded cells [ 22 ]. To determine the effect of ART on the growth rate and proliferation of the bystander cells, the CFSE-labeled cells were co-cultured in a 12-well plate with ART pre-treated Cx43-MCF7 cells.
Results 34
The comparative analysis of Cx43 mRNA expression in MCF7 and Cx43-MCF7 cells was analyzed using real-time PCR. Such a difference in the G1 population suggests that the Cx43-MCF7 cells were more sensitive to G1 arrest after treatment. The generation of ROS in MCF7 and Cx43-MCF7 cells was assessed by measuring DCF fluorescence using a flow cytometer.
To test the above possibility, the MCF7 or Cx43-MCF7 cells were labeled with CFSE dye and.
Discussions 49
In this study, it was deduced that the IC50 of ART treatment on MCF7 cells was significantly reduced when Cx43 was overexpressed in the cells. Therefore, the data suggest that Cx43 may be involved in the sensitization of Cx43-MCF7 cells to ART treatment by controlling the expression of Skp2, which increases the level of tumor suppressor proteins, p27 Kip1 and p21Cip1. Based on the observation of an increase in the expression of p27 Kip1 and p21Cip1, cell cycle profiling of the treated cell population was performed.
Moreover, the addition of NAC and catalase in the coculture medium abolished the bystander effect of ART.
Pan, Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action, Mol. Jiang, Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer, Cancer. Park, The Role of Heme and Mitochondria in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Antimalarial Artemisinin, J Biol Chem.
Park, Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds, J Biol Chem.
4-phenylbutyrate Manifests Synergistic Interaction with
Introduction 61
A large body of evidence suggests that inactivation of HATs and aberrant expression of HDACs results in suppression of several antiproliferative genes in cancer cells, which further mediate tumorigenesis or progression [ 1 , 2 ]. This further regulates the expression of various tumor-associated genes involved in cell cycle control and apoptosis of cancer cells [5]. Increased GJ communication was associated with increased chemosensitivity of cancer cells.
In the current study, a combination therapy involving 4-PB & ART was designed and its efficacy investigated both in vitro and in vivo.
Materials and Methods 63
After the end of the treatment period, the cells were stained with PE annexin V and 7-AAD. Cytofluorimetric analysis of cells after synchronization and treatment with ART and/or 4-PB showed that MCF-7 cells were progressively blocked in the G0/G1 phase of the cell cycle. A decrease in the weight of the mice was an indicator of a decrease in tumor volume.
The reduction in the volume of the tumor after treatment with ART and/or 4-PB, both visually and by weighing the mice, was found.
Introduction 95
Although the CD/5-FC system has demonstrated its efficacy in many clinical trials, its use in therapy has been limited [16-18]. Yeast cytosine deaminase (yCD) has been shown to have a better affinity for 5-FC compared to bCD. An attempt was made to generate a stable bCD mutant by random mutagenesis that has a slightly improved affinity for the prodrug, 5-FC [ 20 ].
The results showed that this mutant significantly increased the therapeutic efficacy of CD/5-FC-mediated suicide gene therapy on A549 cells and has the potential to emerge as a substitute for wtCD.
Materials and Methods 97
Cells were allowed to adhere overnight and transfection was performed in serum-free medium. The cells were then thoroughly washed with fresh PBS and visualized under a fluorescence microscope (Nikon Eclipse Ti-U, Japan; excitation filter of 480/15 nm for AO and 540/25 nm for EtBr, respectively). Next, the stained cells were analyzed by flow cytometry (FacsCalibur, BD Biosciences, NJ) at 10,000 events each.
Furthermore, the extent of apoptosis in the cells was analyzed using a flow cytometer (CytoFLEX, Beckman Coulter).
Results and Discussions 101
Most of the cancer cells including MCF-7 cells are devoid of GJIC and therefore the drugs are in the tumor microenvironment. The decrease in cell viability of the spheroids was due to the uptake of 4-PB-bound NCs. Calcein AM- and PI-based double staining revealed the morphological evidence of the cell viability.
The expression of Cx43 protein in conjunction with the CD/F186W gene enhanced the activity of the suicide gene therapy mediated by CD and F186W mutant.
Conclusion 117
Silver nanocluster based nanocarrier enhances the activity
Introduction 123
From this point of view, nanocarrier (NC)-mediated drug delivery offers a potential tool as it exhibits high efficiency due to its small size and tunable physicochemical properties. However, the main obstacle facing any conventional drug treatment is access to the tumor site, mainly due to the complex cellular organization in the body. Ag can induce the production of ROS inside the cell at low doses, and due to the minimal expression of the antioxidant enzyme in cancer cells, Ag has been found to have maximum toxic effects on cancer cells and minimal effects on normal cells, demonstrating its applicability in a biological system [5].
In this study, Ag-based NCs were used for stable delivery of 4-PB in cells and in a spheroid model.
Materials and Methods 125
Cells were exposed to different concentrations of NCs or 4-PB-NCs or PBS (control) for 48 h. Cells were then washed thoroughly with fresh PBS and visualized under a confocal microscope (Zeiss, LSM 880). Next, flow cytometric assessment was done, for which cells were treated with NC for 4 h.
Cells were then stained with dichlorofluorescin diacetate (DCFDA), and data were then acquired using the CytoFLEX flow cytometer (Beckman Coulter).
Results and Discussions 129
This may be due to the additive cytotoxicity effect of 4-PB alone and NC. After treatment with NCs-4-PB, a significant decrease in cell viability of spheroids was observed compared to 4-PB and NCs alone. A confocal microscopic image of the spheroids revealed NC uptake after 4 h of incubation.
Images obtained with a confocal microscope showed a phase-contrast image of the spheroid (a) and the luminescence of the NCs taken up by the spheroids (b).
Conclusion 139
The importance of this study, highlights of the work that was done as well as the aspect of the possible clinical implications of this research work were investigated in this. The expression of Cx43 in MCF-7 cells established a functional GJIC and demonstrated its potency via GJ dependent as well as independent pathways. Through a well-orchestrated interplay between GJ-dependent as well as GJ-independent action of Cx43, a new perspective of the effective ART treatment was discovered in conjunction with Cx43 overexpression in MCF7 cells.
Future perspectives include studying the efficacy of this system in in vivo and clinical samples.
Concluding Remarks and Future Outlook 141
Concluding Remarks 143
Future Outlook 145
