Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2.

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells' expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

Preparation of iron oxide mesoporous magnetic microparticles as novel multidrug carriers for synergistic anticancer therapy and deep tumor penetration.

The preparation of mesoporous iron oxides with controllable physiochemical properties for effective therapeutic drug delivery remains a formidable challenge. Herein, iron oxide mesoporous magnetic microparticles (IO-MMMs) were prepared by a modified reverse hard-templating approach using, for the first time, acid-prepared mesoporous spheres (APMS) as the hard silica template. The obtained mesostructures exhibited remarkably high surface area and large pore volumes (SBET = 240 m2/g and Vpore = 0.55 cm3/g), controllable average sizes, generally uniform morphologies, and excellent biocompatibilities, allowing them to achieve optimal drug release in cancer cells and tumor tissues. IO-MMM carriers were able to co-load high amounts of hydrophilic chemotherapeutic drugs (Dox or Daun) and/or hydrophobic hormonal anticancer drugs (Tam), and release them sustainably in a pH-dependent manner, utilizing the fluorescence of Daun to real-time trace the intracellular drug distribution, and employing Daun/Tam to treat cancer by combined chemo/hormonal therapy. Cytotoxicity assays against different types of cancerous cells showed that the combinatory Daun/Tam@IO-MMM formulation significantly reduced the viability of metastatic MCF7 and KAIMRC1 breast as well as HCT8 colorectal cancer cells, with the least potency towards non-cancerous normal primary cells (up to 10-fold). Electron, flow, and live confocal microscopy imaging confirmed that the loaded vehicles were successfully and differentially uptaken by the different tested cells, gradually releasing their payloads, and causing apoptotic cell death. Importantly, compared to free drugs, Daun/Tam@IO-MMMs displayed enhanced drug accumulation in patient breast primary tumor tissues, deeply penetrating into the tumor region and killing the tumor cells inside. The designed carriers described here, thus, constitute a novel promising magnetic mesoporous smart system that entraps different kinds of drugs and release them in a controlled manner for combinatorial chemo/hormonal cancer theranostics. This multifactorial platform may open new avenues in cancer therapy as efficient synergistic antitumor system through overcoming limitations of conventional cancer therapy.

Characterization and hepatoprotective evaluation of sesquiterpenes and diterpenes from the aerial parts of Juniperus sabina L.

Previously, we reported on the hepatoprotective activity of the total extract of Juniperus sabina L. against CCl4 induced liver toxicity in experimental animals. Biologically directed phytochemical study was conducted to identify the active compounds. Male Wistar rats and the standard drug silymarin were used in the study. Hepatoprotective activity was evaluated via serum biochemical parameters such as aspartate amino transferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Tissue parameters including non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP) were also determined. Histopathological study was conducted utilizing Mayer's hematoxylin stain, Periodic Acid Schiff - Hematoxylin (PAS-H) and Masson trichrome technique on light microscope. Electron microscope images were also generated for the study. The activity of the total extract was trapped to the petroleum ether fraction after liquid-liquid fractionation where 51% reduction in the levels of AST, bilirubin and 44% in the levels of ALT were observed. Chromatographic purification of the petroleum ether fraction resulted in the isolation of nine compounds namely: trans-calamenene (1), cadalene (cadalin) (2), epi-cubenol (3), manool (4), calamenene-10ß-ol (5), calamenene-10α-ol (6), 4-epi-abietic acid (7), sandaracopimaric acid (8) and isopimaric acid (9). Compounds 1-3, 5 and 6 are belonging to cadinane sesquiterepenes, while compounds 4, 7-9 were of diterpene skeleton. The major compounds were tested for their hepatoprotective effect. Compounds 3 showed marked improvement in the levels of AST and ALT, compound 4 was effective in improving the levels of AST, ALT, GGT, ALP and bilirubin, while compound 7 showed significant improvement in GGT, ALP and bilirubin levels.

Evaluation of the hepatoprotective effect of combination between hinokiflavone and Glycyrrhizin against CCl4 induced toxicity in rats.

Liver diseases are one of the fatal syndromes due to the vital role of the liver. Most of the effective treatment of liver conditions are of natural origin. Silymarin (SI) is the standard drug used for treatment of impaired liver functions. Two natural compounds possessing promising liver protection and with different chemical structures namely; the bioflavonoid hinokiflavone (HF) isolated from Junipers phoenicea family Cupressaceae and the sweet saponin Glycyrrhizin (GL) present in Glycyrrhiza glabra (liquorice) were selected for the current study. Since the two compounds are of different nature, they may act by different mechanisms and express synergistic effect. Combination of the two compounds using to dose levels were challenged with single doses of HF, GL and SI as well. The comparison was monitored via measuring serum biochemical parameters including, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltranspeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin, tissue parameters such as MDA, NP-SH and TP, histopathological study using light and electron microscope. Protective effect on kidney was also monitored histopathologically and biochemically through observing the levels of LDH, creatinine, creatinine-kinase, urea and uric acid. The combinations of HF and GL showed protective effect more than the used single doses of HF and GL alone. However, SI was superior to the used combination in the two used doses in all the measured parameters. The liver and kidney cells appearance under normal and electron microscope showed that SI treated groups showed almost normal cells with slight toxic signs. Cells from group treated with the higher doses of the combination of HF and GL showed slight signs of intoxication under light and electron microscope indicating good level of protection. Although the combination of HF and GL expressed good protection in the higher dose, however, the combination did not exceed the protective effect of SI.