A Combined Recombinant Retroviral-Based Therapy for Colon Cancer in the Murine Model Using Anti-Tumorigenic and Anti-Angiogenic Agents

Anti-tumorigenic gene therapy offers many benefits over than conventional therapy i.e., chemotherapy or radiotherapy including less toxic side effects, rapid delivery and more sensitive compared to target-based drugs. Anti-tumorigenic agents alone were not proven to be effective in promoting the delay or regression of tumor mass in vivo due to low therapeutic gene transfer efficiency and the formation of new blood vessels in tumors. In addition, the blood vessels are one of the major factors that reduce the effectiveness of anti-tumorigenic agents. Consequently, a new therapeutic approach that combined both anti-tumorigenic and anti-angiogenic agents was established and proven to provide superior and beneficial synergistic effect. In the present study, a complete open reading frame (ORF) encoding endostatin protein was RNA extracted from a Balb/c mice liver tissue and transcribed into cDNA. The gene was then cloned into TOPO vector prior to a retroviral vector, pMSCVneo. Upon cloning and sequencing of the full length of the gene, it was revealed that the complete ORF contained 550 nucleotides, by which the size similar to that of established endostatin. While, anti-tumorigenic gene; VP3 was obtained from previous study (generous gift from Professor Mohd Azmi Mohd Lila) and verified by PCR approaches. The PCR amplification and restriction endonuclease analysis showed that the gene product was convenient and similar to the expected size. The gene obtained was also 98% homology to that the references strain, Cux-1 by DNA sequencing analysis. Both recombinant constructs were transfected into PT67 cells and the transiently produced virus used to treat Balb/c mice with established colon tumors. The method of treatments was given in either as single modality or combined treatment with standardized of tumor size in each mice group. Here, we also revealed a viral delivery vehicle named 'retroviral vector’ to deliver the both anti-tumorigenic and anti-angiogenic agents into the tumor mass to achieve the effects of apoptosis. Furthermore, the retroviral-mediated gene therapies also showed no signs of adverse effect. Based on PCR approaches, the recombinant retrovirus particles were disseminated into the bloodstream and finally presented in various tissues but only expressed their therapeutic genes in tumors. As a result, the risk of undesirable characteristics of cancer gene therapy could be avoided in non-target tissues. The differential activities of VP3 and endostatin were investigated by the use of the immunohistochemistry analysis such as PCNA and vWF. The apoptosis activities upon expression of VP3 and endostatin were further studied by quantifying and identifying biochemical changes and number of affected cells due to apoptosis. Upon TUNEL and DNA fragmentation assays, typical apoptotic patterns were observed, to include formation of apoptotic bodies and visible DNA ladders, as early as 48 hours after treatments. Since necrotic cells could be stained positively by TUNEL, a flow cytometry assay was developed for identifying and quantifying dead or dying cells in mode of cell death. The apoptosis percentage including early and late apoptotic quadrants showed that the combined tumor treatment was higher than VP3 and endostatin only. The effect of VP3 and endostatin protein expression in tumor and vital organ tissues were also confirmed upon examination by inverted microscopy analysis with H&E-stained sections. In addition, upon electron microscopic examination, typical morphological apoptotic features were observed including intact membranes and organelles, cell blebbing as well as condensed nuclear membranes in single- and combined-treated tumors not in other vital organ tissues. Interestingly, based on these constructive findings the combined treatment noted better responses in tumor bearing mice rather than single treatments. In conclusion, this study showed that the combined approach was more promising, effective and good governance on delay tumor growth and inducing apoptosis than introducing VP3 or endostatin alone. The combination treatment also offers potential benefits in control of tumorigenesis, and thus deserves further research as a preferred approach in cancer gene therapy

Blood-brain barrier derangement after electrical brain stimulation

Noninvasive brain stimulation methods, including repetitive transcranial magnetic stimulation and transcranial direct current stimulation (tDCS), have received considerable attention in recent years for use in the study and treatment of neurological conditions. Of these methods, tDCS is considered particularly promising due to its ease of use and ability to confer polarity-dependent effects on brain excitability, making it an excellent option for clinical treatment of neurological and psychiatric diseases. While generally regarded as safe when following standard protocols, the effects of tDCS on cerebral blood vessels and blood-brain barrier (BBB) functions remain poorly understood. Here, we provide an overview of tDCS in the context of BBB function, summarize the current literature, and discuss implications for future research. To date, no alterations or damage to the BBB have been reported after weak tDCS stimulations in human subjects; however, some animal studies have reported alterations to BBB function following increased tDCS intensity, with inconsistencies in the effective tDCS polarity used to produce these BBB disruptions between studies. Further research will be necessary to evaluate the effects of tDCS on the BBB under various conditions. Finally, we discuss the potential of tDCS for enhancing drug delivery to the central nervous system, which may become possible as we refine our understanding of the effects of tDCS on BBB permeability

HIF-1 ACTIVATION AND INFLAMMATORY RESPONSES TO HYPOXIA

Acute hypoxia is a significant physiological danger during high-altitude flying and military aircraft missions. The human brain requires a constant supply of oxygen to function properly, and is susceptible to settings with low availability of air oxygen. Hypoxia can influence inflammatory signalling, and both central and systemic responses can activate HIF pathway genes. HIFs are critical molecules that regulate inflammation andhypoxia, ensuring appropriate cell function and survival. Hypoxia is the condition in which insufficient oxygen reaches the body\u27s tissues. It can be caused by a decrease in partial oxygen pressure (PO2) in the environment, problems with breathing and/or oxygen transport, or the inability of tissues to utilise oxygen. Different organs are hypoxic due to differences in tissue oxygen tensions, which are determined by differences in aerobic metabolism. Extremely hypoxic individuals have the most dramatic systemic and neurological adaptations to persistent hypoxia. In this review, we provide an overview of central and systemic responses to hypoxia and discuss the activation of HIF-1 pathway

Quantification of infectious recombinant murine MSCV-based retroviruses using PCR approach

The current available molecular method to detect the retroviral-mediated gene expression is reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and not sensitive. In this paper, we performed a real-time PCR assay to quantify retroviral mediated gene expression in the supernatant of infectious recombinant virus. We documented an optimized methodology for accurate and reproducible quantification of retrovirus-based gene expression which enables rapid and quantitative determination of sample gene expression. We have also compared the performance of the assay with our routine RT-PCR assay. RNA was extracted from infectious recombinant retrovirus at 48 h post-infection and assayed for VP3 gene. The standard curve from linear DNA standards showed high sensitivity and good linearity (R2 = 0.9902 for VP3 standards graphs), ranged from 102 to 108 copies of comparable accuracy to current quantification real-time PCR methods. The present study presumes a copy number of transgene expression to equal a molecule of infectious recombinant virus particle. Another advantage, this concept could determine vector stability by comparison of infectious particles, total particles and particles with RNA. In comparison to gel-based RTPCR, we conclude that real-time PCR as a better approach in gene expression quantification

Current techniques in reprogramming cell potency

The first successful attempt to reprogram somatic cell into embryonic-like stem cell was achieved on 2006. Since then, it had sparked a race against time to bring this wonderful invention from bench to bedside but it is not easily achieved due to severe problems in term of epigenetic and genomic. With each problem arise, new technique and protocol will be constructed to try to overcome it. This review addresses the various techniques made available to create iPSC with problems hogging down the technique

Comparative study of antioxidant level and activity from leaf extracts of Annona muricata Linn obtained from different locations

Annona muricata Linn possesses an anti-tumorigenic effect towards cancer. Several of its bioactive components have already been assessed in previous findings. However, none of the previous studies actually addressed the important consideration of the association between cultivation area of this medicinal plant and its bioactive compounds/antioxidants. In this study, the antioxidant level and antioxidant activity of 19 Annona muricata collected from different locations were evaluated by phenolic and flavonoid assays together with Oxygen Radical Absorbance Capacity (ORAC), Ferric Reducing Ability of Plasma (FRAP) and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assays. M1 was found to have an attractive antioxidant profile as it had the highest content of phenolics (73.2 µg/mL GAE) and flavonoids (191.4 µg/mL CE) and also the highest antioxidant capacity in ORAC assay (254.7 µM). Additionally, it had a favourably high ferric ion reducing capacity (15.55 µM Fe2+/µg) and the best free DPPH-radical scavenging activity (IC50=143.5 µg/mL). On the contrary, R1 showed the lowest level of phenolics with a GAE value of 21.92 µg/mL, ranked second lowest in flavonoid content (65.42 µg/mL CE), and it had the least antioxidant capacity in ORAC (94.66 µM), FRAP (4.17 µM Fe2+/µg) and DPPH assays (1597 µg/mL), making it the least desirable antioxidant source. Based on this finding, it was concluded that Annona muricata Linn had varied antioxidant levels and activity regarding its cultivation area; hence, it would be a guide in the selection of potential candidates for natural antioxidants in phytopharmacy

Apoptosis and tumour cell death in response to pro-apoptotic gene

The process of cell death, or apoptosis, is commonly defined by its distinct morphological characteristic. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, the cellular antiviral response pathway, embryonic development and chemical-induced cell death. In this study, retroviral vector was utilized as the gene delivery vehicles and the tumour-specific viral death effector VP3 as pro-apoptotic agent for malignant colon cancer cells treatment. Here, pro-apoptotic gene inducing apoptosis in CT26 colon tumour cells is reported. In addition, the activation of a typical apoptotic programme was observed in response to this therapeutic agent. An increased number of colon tumour cells suffered apoptosis upon treatment with pro-apoptotic agent. As a result, these tumour cells displayed loss of membrane asymmetry and impermeability, cell shrinkage, nuclear condensation and DNA fragmentation under H&E images. In the presence of VP3, TEM also confirmed that VP3-treated CT26 tumour cells showed apoptotic features. These results suggested that VP3 was a potential pro-apoptotic agent and the apoptosis induced by VP3 was a key anti-tumour mechanism

Anti-cancer effect of Annona Muricata Linn Leaves Crude Extract (AMCE) on breast cancer cell line

Background: Annona muricata Linn which comes from Annonaceae family possesses many therapeutic benefits as reported in previous studies and to no surprise, it has been used in many cultures to treat various ailments including headaches, insomnia, and rheumatism to even treating cancer. However, Annona muricata Linn obtained from different cultivation area does not necessarily offer the same therapeutic effects towards breast cancer (in regards to its bioactive compound production). In this study, anti-proliferative and anti-cancer effects of Annona muricata crude extract (AMCE) on breast cancer cell lines were evaluated. Methods: A screening of nineteen samples of Annona muricata from different location was determined by MTT assay on breast cancer cell lines (MCF-7, MDA-MB-231, and 4 T1) which revealed a varied potency (IC50) amongst them. Then, based on the IC50 profile from the anti-proliferative assay, further downward assays such as cell cycle analysis, Annexin V/FITC, AO/PI, migration, invasion, and wound healing assay were performed only with the most potent leaf aqueous extract (B1 AMCE) on 4 T1 breast cancer cell line to investigate its anti-cancer effect. Then, the in vivo anti-cancer study was conducted where mice were fed with extract after inducing the tumor. At the end of the experiment, histopathology of tumor section, tumor nitric oxide level, tumor malondialdehyde level, clonogenic assay, T cell immunophenotyping, and proteome profiler analysis were performed. Results: Annona muricata crude extract samples exhibited different level of cytotoxicity toward breast cancer cell lines. The selected B1 AMCE reduced the tumor’s size and weight, showed anti-metastatic features, and induced apoptosis in vitro and in vivo of the 4 T1 cells. Furthermore, it decreased the level of nitric oxide and malondialdehyde in tumor while also increased the level of white blood cell, T-cell, and natural killer cell population. Conclusion: The results suggest that, B1 AMCE is a promising candidate for cancer treatment especially in breast cancer and deserves further research as an alternative to conventional drugs while also stressed out the selection of soursop sample which plays a significant role in determining its potential therapeutic effect on cancer

Tissue distribution of intramuscularly and intratumouraly administered DNA plasmid harbouring apoptotic gene in mice

This study investigated the bio-distribution and persistence of plasmid DNA following intramuscular and intratumoural administration in a mice model. Validated quantitative method (real-time qPCR) was used to quantify plasmid distribution in the tissue samples collected at 15 min, 1 h, 24 h and 1 week after administration of 100 μg (1.5 x 1013 copies) of naked plasmids. Plasmids remained in the circulating blood (3.6 ± 2.2 x 102copies/500 ng gDNA) and injected muscle (2.8 ± 1.1 x 105 copies/500ng gDNA) for up to 1 week post administration. Plasmids were also detected in opposite muscle, lung, kidney, spleen, lymph nodes, liver and heart only 1 h post-injection or more. After 2 weeks of treatment, plasmids were retained solely in the tumor mass. These results suggest the presently used recombinant DNA plasmid was benefited with its early transgene expression characteristic which could release the anti-cancerous effect within short dwelling time

The Regulatory Role of MicroRNAs in Breast Cancer

MicroRNAs (miRNAs) are small non-coding RNA molecules which function as critical post-transcriptional gene regulators of various biological functions. Generally, miRNAs negatively regulate gene expression by binding to their selective messenger RNAs (mRNAs), thereby leading to either mRNA degradation or translational repression, depending on the degree of complementarity with target mRNA sequences. Aberrant expression of these miRNAs has been linked etiologically with various human diseases including breast cancer. Different cellular pathways of breast cancer development such as cell proliferation, apoptotic response, metastasis, cancer recurrence and chemoresistance are regulated by either the oncogenic miRNA (oncomiR) or tumor suppressor miRNA (tsmiR). In this review, we highlight the current state of research into miRNA involved in breast cancer, with particular attention to articles published between the years 2000 to 2019, using detailed searches of the databases PubMed, Google Scholar, and Scopus. The post-transcriptional gene regulatory roles of various dysregulated miRNAs in breast cancer and their potential as therapeutic targets are also discussed