الفهرس 
Introduction and Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma (HCC) is a universal problem and its epidemiological data showed variation from place to place. Egypt ranks the third and 15th most populous country in Africa and worldwide, respectively. Typically, HCC is often diagnosed at an advanced stage, and thus many patients are not eligible for the curative therapies. Therefore clear determination of HCC risk factors would be very helpful for well-designed strategies for HCC prevention. Generally, prevention of HCC is based on early prevention of HCC risk factors (primary prevention), treatment of risk factors at an early stage (secondary prevention), and decreasing relapse after successful curative treatment (tertiary prevention).
Many systemic cytotoxic chemotherapy drugs are used in HCC treatment as single agents, e.g., cisplatin, doxorubicin, 5-fluorouracil, or as combined regimens. Although it is effective in killing the cancer cells, doxorubicin also exerts undesirable cytotoxic effects on normal cells in various organs including the heart, brain and kidney, and elicits dose-dependent acute and chronic toxic side effects. In terms of the severe resistance to drugs in HCC, systemic chemotherapy can rarely achieve the anticipated effect, given that HCC patients with advanced stage can hardly benefit from surgery. Traditional chemotherapy is still used in HCC as a palliative treatment modality to attenuate the symptoms caused by HCC and improve the patients’ life quality. Currently, there is a paradigm shift in HCC treatment by the introduction of immune checkpoint inhibitors, in addition to molecular- targeted therapies, which are limited to patients who can afford the cost.
Despite the advances in HCC treatment, it remains one of the most difficult cancers to treat, and recurrence of HCC remains a major problem, reaching an incidence of more than 70% at 5 years. In addition to chemo-therapy, great progress has been made in radiotherapy for treatment of HCC, but it has also a limited role due to resistance. Recently, some new factors, such as miRNA, the immunosuppressive microenvironment and signaling pathways that make resistance more complex have been identified.
Cell cycle control genes are frequently mutated in cancer cells, which usually display higher rates of proliferation than normal cells. Mitosis, a critical and highly orchestrated event in the cell cycle. Errors in the choreography of this event may lead to uncontrolled proliferation, aneuploidy and genetic instability culminating in cancer development. Considering the central role of phosphorylation in mitotic checkpoints, spindle function, and chromosome segregation, it is not surprising that several mitotic kinases have been implicated in tumorigenesis.
Many drugs that disrupt mitosis have been studied because they induce cell cycle arrest and tumor cell death. These antitumor small molecule compounds are referred to as antimitotics. These drugs generally target specific components of mitotic regulation that are mainly or exclusively expressed during cell division, such as kinases, motor proteins and multiprotein complexes. Such compounds are now in preclinical studies and clinical trials, and many are products or derivatives from natural sources.
The aurora kinases are a collection of highly related and conserved serine-threonine kinases that function as key regulators of mitosis and multiple signaling pathways. They are involved in some checkpoint regulation pathways including spindle assembly checkpoint (SAC), alignment of metaphase chromosomes and chromosomal biorientation. Overexpression of aurora kinases is generally detected in a wide variety of human cancers.
In addition, mitotic regulators including INCENP, survivin, CENP-A and MCAK are mainly expressed during cell division. They are involved in spindle function, chromosome segregation, phosphorylation of substrates involved in chromosome condensation, correction of erroneous kinetochore-microtubule attachments, activation of SAC and cytokinesis. Alterations in aurora kinases and mitotic regulators signaling are associated with mitotic errors and may lead to cancer, which make them ideal drug target candidates for antitumor therapies.
Aloin, a natural phytoanthracycline extracted from the leaf exudates of Aloe vera, has been previously reported to possess a cytotoxic effect against different cancer cell lines. The dose-dependent cytotoxic action of aloin was mediated due to multiple modes of action, including repressed protein expression of topoisomerase llα and cyclin Bl, enhanced p53 overexpression, increased proportion of the cells cycling at a higher ploidy level (> G2M). The polypolidization indicates that aloin does not inhibit the initiation of DNA synthesis and that cells replicated a full complement of DNA but had a difficulty in M phase.
The present study aimed at investigating the in vitro chemosensitivity of aloin against a human non tumorigenic hepatocellular carcinoma cell line (HepG2), compared to an anthracycline analog, doxorubicin. The aim was furtherly extended to explore the alterations in aurora kinases and some mitotic regulators in HepG2 cells following exposure to the half maximal inhibitory concentrations (IC50) of aloin and doxorubicin.
The HepG2 cells was incubated with multiple increasingconcentrations of aloin (50-500 μg/ml) for 24 and 72 h or doxorubicin (0.5-12 μg/ml) for 72 h. The cytotoxic effects of aloin and doxorubicin were evaluated by MTT and clonogenic assays, from which IC50 values were calculated and then used to assess the expression of genes involved in the regulation of the mitotic machinery in HepG2 cells.
Results obtained are summarized as follows:
Exposure of HepG2 cells to aloin for 24 and 72 h showed a dose-dependent reduction in the percentage of cell viability. The brief exposure regimen of HepG2 cells to the tested aloin concentrations (50-500 μg/ml) was not enough to induce a 50% inhibition of cellular growth, whereas the continuous exposure regimen was sufficient to calculate the IC50 value (406.6 μg/ml).
Aloin at doses 100-500μg/ml significantly inhibited the tumor colonies formation in HepG2 cells in both brief and prolonged exposure regimens.
On the other hand, all tested doxorubicin doses (0.5-12 μg/ml) showed a significantly dose-dependent reduction in the percentage of cell viability after incubation for 72 h (IC50 value of 1.022 μg/ml) and inhibition of colony formation at continuous exposure.
The continuous exposure of HepG2 cells to IC50 value of aloin significantly down-regulated mRNA expression of aurora kinase A and MCAK genes, as well as survivin and INCENP proteins expression, and by contrast up-regulated the mRNA expression of aurora kinase B gene.
Similarly, the continuous exposure of HepG2 cells to IC50 value of doxorubicin caused a significant down-regulation in the mRNA expression of aurora kinase A and MCAK genes, as well as survivin, INCENP and CENP-A proteins expression, while aurora kinase B gene expression was significantly up-regulated.