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Improved DNA delivery using invasive E. coli DH10B in human cells by modified bactofection method

Sultana A, Tiash S

J Control Release, 2021 04 10;332:233-244.
PMID: 33561481 DOI: 10.1016/j.jconrel.2021.02.004

E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector's efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector's gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.
siRNAs targeting multidrug transporter genes sensitise breast tumour to doxorubicin in a syngeneic mouse model

Tiash S, Chowdhury EH

J Drug Target, 2019 03;27(3):325-337.
PMID: 30221549 DOI: 10.1080/1061186X.2018.1525388

Chemotherapy, the commonly favoured approach to treat cancer is frequently associated with treatment failure and recurrence of disease as a result of development of multidrug resistance (MDR) with concomitant over-expression of drug efflux proteins on cancer cells. One of the most widely used drugs, doxorubicin (Dox) is a substrate of three different ATP-binding cassette (ABC) transporters, namely, ABCB1, ABCG2 and ABCC1, predominantly contributing to MDR phenotype in cancer. To silence these transporter-coding genes and thus enhance the therapeutic efficacy of Dox, pH-sensitive carbonate apatite (CA) nanoparticles (NPs) were employed as a carrier system to co-deliver siRNAs against these genes and Dox in breast cancer cells and in a syngeneic breast cancer mouse model. siRNAs and Dox were complexed with NPs by incubation at 37 °C and used to treat cancer cell lines to check cell viability and caspase-mediated signal. 4T1 cells-induced breast cancer mouse model was used for treatment with the complex to confirm their action in tumour regression. Smaller (∼200 nm) and less polydisperse NPs that were taken up more effectively by tumour tissue could enhance Dox chemosensitivity, significantly reducing the tumour size in a very low dose of Dox (0.34 mg/kg), in contrast to the limited effect observed in breast cancer cell lines. The study thus proposes that simultaneous delivery of siRNAs against transporter genes and Dox with the help of CA NPs could be a potential therapeutic intervention in effectively treating MDR breast cancer.
Passive Targeting of Cyclophosphamide-Loaded Carbonate Apatite Nanoparticles to Liver Impedes Breast Tumor Growth in a Syngeneic Model

Tiash S, Chowdhury ME

Curr Pharm Des, 2016;22(37):5752-5759.
PMID: 26864311

Despite being widely used for treating cancer, chemotherapy is accompanied by numerous adverse effects as a result of systemic distribution and nonspecific interactions of the drugs with healthy tissues, eventually leading to therapeutic inefficacy and chemoresistance. Cyclophosphamide (Cyp) as one of the chemotherapeutic pro-drugs is activated in liver and used to treat breast cancer in high dose and in combination with other drugs. In an attempt to reduce the off-target effects and enhance the therapeutic efficacy, pH-sensitive carbonate apatite nanoparticles that had predominantly and size-dependently been localized in liver following intravenous administration, were employed to electrostatically immobilize Cyp and purposely deliver it to the liver for activation. Cyp-loaded particles formed by simple 30 min incubation at 37ºC of the DMEM (pH 7.4) medium containing CaCl2 and Cyp, enhanced in vitro cytotoxicity at different degrees depending on the cell types. The size of the particles could be tightly controlled by the amount of CaCl2 required to prepare the particles and thus the bio-distribution pattern inside different organs of the body. Unlike the small particles (~ 200 nm), the large size particles (~ 600 nm) which were more efficiently accumulated in liver, significantly reduced the tumor volume following intravenous injection in 4T1-induced murine breast cancer model at a very low dose (0.17 mg/Kg) of the drug initially added for complex formation, thus shedding light on the potential applications of the Cyp-loaded nano-formulations in the treatment of breast cancer.
Knockdown of ROS1 gene sensitizes breast tumor growth to doxorubicin in a syngeneic mouse model

Tiash S, Chua MJ, Chowdhury EH

Int J Oncol, 2016 Jun;48(6):2359-66.
PMID: 27035628 DOI: 10.3892/ijo.2016.3452

Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.
Carbonate apatite nanoparticles carry siRNA(s) targeting growth factor receptor genes egfr1 and erbb2 to regress mouse breast tumor

Tiash S, Kamaruzman NIB, Chowdhury EH

Drug Deliv, 2017 Nov;24(1):1721-1730.
PMID: 29119846 DOI: 10.1080/10717544.2017.1396385

Cancer cells lose their control on cell cycle by numerous genetic and epigenetic alterations. In a tumor, these cells highly express growth factor receptors (GFRs), eliciting growth, and cell division. Among the GFRs, epidermal growth factor receptor-1 (EGFR1) (Her1/ERBB1) and epidermal growth factor receptor-2 (EGFR2) (Her2/ERBB2) from epidermal growth factor (EGF) family and insulin-like growth factor-1 receptor (IGF1R) are highly expressed on breast cancer cells, thus contributing to the aggressive growth and invasiveness, have been focused in this study. Moreover, overexpression of these receptors is related to suppression of cell death and conferring resistance against the classical drugs used to treat cancer nowadays. Therefore, silencing of these GFRs-encoding genes by using selective small interfering RNAs (siRNAs) could be a powerful approach to treat breast cancer. The inorganic pH sensitive carbonate apatite nanoparticles (NPs) were used as a nano-carrier to deliver siRNA(s) against single or multiple GFR genes in breast cancer cells as well as in a mouse model of breast carcinoma. Silencing of egfr1 and erbb2 simultaneously led to a reduction in cell viability with an increase in cell death signal in the cancer cells and regression of tumor growth in vivo.
Methotrexate- and cyclophosphamide-embedded pure and strontiumsubstituted carbonate apatite nanoparticles for augmentation of chemotherapeutic activities in breast cancer cells

Tiash S, Othman I, Rosli R, Chowdhury EH

Curr Drug Deliv, 2014;11(2):214-22.
PMID: 24328684

Most of the classical drugs used today to destroy cancer cells lead to the development of acquired resistance in those cells by limiting cellular entry of the drugs or exporting them out by efflux pumps. As a result, higher doses of drugs are usually required to kill the cancer cells affecting normal cells and causing numerous side effects. Accumulation of the therapeutic level of drugs inside the cancer cells is thus required for an adequate period of time to get drugs' complete therapeutic efficacy minimizing the side effects on normal cells. In order to improve the efficacy of chemotherapeutic drugs, nanoparticles of carbonate apatite and its strontium (Sr(2+))-substituted derivative were used in this study to make complexes with three classical anticancer drugs, methotrexate, cyclophosphamide and 5-flurouracil. The binding affinities of these drugs to apatite were evaluated by absorbance and HPLC analysis and the therapeutic efficacy of drug-apatite complexes was determined by cell viability assay. Carbonate apatite demonstrated significant binding affinity towards methotrexate and cyclophosphamide leading to more cellular toxicity than free drugs in MCF-7 and 4T1 breast cancer cells. Moreover, Sr(2+) substitution in carbonate apatite with resulting tiny particles less than 100 nm in diameter further promoted binding of methotrexate to the nanocarriers indicating that Sr(2+)-substituted apatite nanoparticles have the high potential for loading substantial amount of anti-cancer drugs with eventual more therapeutic effectiveness.
Fulltext Knockdown of PLC-gamma-2 and calmodulin 1 genes sensitizes human cervical adenocarcinoma cells to doxorubicin and paclitaxel

Stanislaus A, Bakhtiar A, Salleh D, Tiash S, Fatemian T, Hossain S, et al.

Cancer Cell Int, 2012 Jun 18;12(1):30.
PMID: 22709569 DOI: 10.1186/1475-2867-12-30

BACKGROUND: RNA interference (RNAi) is a powerful approach in functional genomics to selectively silence messenger mRNA (mRNA) expression and can be employed to rapidly develop potential novel drugs against a complex disease like cancer. However, naked siRNA being anionic is unable to cross the anionic cell membrane through passive diffusion and therefore, delivery of siRNA remains a major hurdle to overcome before the potential of siRNA technology can fully be exploited in cancer. pH-sensitive carbonate apatite has recently been developed as an efficient tool to deliver siRNA into the mammalian cells by virtue of its high affinity interaction with the siRNA and the desirable size distribution of the resulting siRNA-apatite complex for effective cellular endocytosis. Moreover, internalized siRNA was found to escape from the endosomes in a time-dependent manner and efficiently silence gene expression.
RESULTS: Here we show that carbonate apatite-mediated delivery of siRNA against PLC-gamma-2 (PLCG2) and calmodulin 1 (CALM1) genes has led to the sensitization of a human cervical cancer cell line to doxorubicin- and paclitaxel depending on the dosage of the individual drug whereas no such enhancement in cell death was observed with cisplatin irrespective of the dosage following intracellular delivery of the siRNAs.
CONCLUSION: Thus, PLCG2 and CALM1 genes are two potential targets for gene knockdown in doxorubicin and paclitaxel-based chemotherapy of cervical cancer.