Elsevier

International Immunopharmacology

Volume 4, Issue 14, 20 December 2004, Pages 1775-1784
International Immunopharmacology

Review
Aloe–emodin modulates PKC isozymes, inhibits proliferation, and induces apoptosis in U-373MG glioma cells

https://doi.org/10.1016/j.intimp.2004.07.012Get rights and content

Abstract

Aloe–emodin (1,8-dihydroy-3-[hydroxymethyl]-anthraquione) purified from Aloe vera leaves has been reported to have antitumor activity. The objectives of our research were to determine how aloe–emodin regulates the cell cycle, cell proliferation and protein kinase C (PKC) during glioma growth and development. To establish the cell cycle effects of aloe–emodin on brain cells [transformed glia cell line (SVG) and human glioma U-373MG cell line (U-373MG)], cells were treated with either dimethylsulfoxide (DMSO; control) or aloe–emodin (40 μM). Results from flow cytometry demonstrated that aloe–emodin delayed the number of cells entering and exiting DNA synthesis (S) phase in both SVG and U-373MG cells indicating that aloe–emodin may inhibit S phase progression. Assessment of cell viability demonstrated that SVG and U-373MG glioma cell were highly sensitive to aloe–emodin. The aloe–emodin-induced decreased proliferation was sustained at 48–96 h. A PKC activity assay was quantified to establish the role of PKC in aloe–emodin's mode of action. Exposure of SVG and U-373MG glioma cells to aloe–emodin suppressed PKC activity and reduced the protein content of most of the PKC isozymes. We determined that cancer growth inhibition by aloe–emodin was due to apoptosis (i.e., programmed cell death). Taken together, these results support the hypothesis that aloe–emodin represents a novel antitumor chemotherapeutic drug.

Introduction

Considerable attention has been given recently to the possibility of utilizing aloe–emodin (1,8-dihydroy-3-[hydroxymethyl]-anthraquione) as a chemotherapeutic drug for the treatment of various types of cancers. Aloe–emodin has been reported to have antitumor activity in neuroectodermal tumors [1], neuroblastomas [1], lung carcinoma [2], [3], leukemia cells [4], [5], promyeloleukemic HL-60 cells [6], hepatoma cells [7] and in a Merkel carcinoma cell line [8]. Aloe–emodin's mechanism of action involves apoptosis (programmed cell death) as judged by DNA fragmentation and morphological features [1], [2], [3], modulation of Bcl-2, Bax and Fas family of proteins [2], [7], increased cytochrome c [2], [3], activation of caspase-3 [2], [3], caspase-8 [2] and caspase-9 [2], and increased p53 and p21 proteins [7]. Additionally, aloe–emodin's mechanism of induced apoptosis appears to involve protein kinase C (PKC) [3]. PKC is a family of 11 known isozymes which are found in varying ratios in the cytosolic and membrane fractions of cells, depending on the type of tissue and its physiological state [9]. PKC isozymes can be classified into three groups. Group I includes Ca2+-dependent isozymes: cPKC-α, cPKC-βI, cPKC-βII and cPKC-γ. Isozymes in group II, nPKC-ɛ, nPKC-δ, nPKC-η and nPKC-θ are Ca2+ independent. Group III includes the atypical PKC: aPKC-ί [10], aPKC-ζ and aPKC-μ (protein kinase D) which are phospholipid dependent. PKC regulates cellular functions, metabolism and proliferation by phosphorylating proteins in response to transmembrane signals from hormones, growth factors, neurotransmitters and pharmacological agents. Thus, agents that inhibit PKC isozymes may block tumor progression. Information on the effects of aloe–emodin on cell proliferation and PKC isozymes is important because PKC overproduction has been linked with the rapid growth rate of gliomas [11], [12].

In this investigation, we evaluated aloe–emodin's antitumor effect on the cell cycle, cell proliferation, PKC activity, PKC isozyme content and apoptosis in transformed glia cell line (SVG) [13] and human glioma U-373MG cell line (U-373MG) which are a highly lethal brain tumor cell line.

Section snippets

Aloe–emodin

Aloe–emodin (1,8-dihydroy-3-[hydroxymethyl]-anthraquione) was purchased from Sigma (catalogue number A7687; St. Louis, MO).

Cell culture

The U-373MG and SVG transformed cell lines (glial cells transformed with the human papovavirus JCV; [13]) were obtained from the American Tissue Culture Collection (Rockville, MD). Cells were seeded (1×106) and grown as monolayers in 75-cm2 flasks containing 90% Dulbecco's modified Eagle's media (DMEM), 10% fetal calf serum (FCS), 2 mM l-glutamine, 4.5 gm/l glucose and

Aloe–emodin delays DNA synthesis (S) phase progression

To establish the cell cycle effects of aloe–emodin on SVG transformed glial cells and U-373MG glioma cells, we used confluent cultures to obtain cells with a high quiescence/Gap 1 (G0/G1) cell population and attempted a reversible G0/G1 arrest by serum starvation. Cells were grown to confluence and serum starved for 48 h. The cell cycle was initiated by serum addition in combination with either dimethylsulfoxide (DMSO; vehicle control) or aloe–emodin (40 μM; dissolved in DMSO). This

Discussion

Our experiments provide further support for aloe–emodin as a potential anticancer chemotherapeutic drug. The data presented here established that aloe–emodin delays S phase progression, inhibits SVG and U-373MG cell proliferation, decreases PKC activity, reduces PKC isozyme protein content (with the exception of PKC-ι) and induces apoptosis, as judged by a reduction in PARP, cleavage of caspase 7 and a decrease in survivin. Other laboratories have published the chemopreventive [30] and

Acknowledgements

We acknowledge the important contribution of the Flow Cytometry Core, H. Lee Moffitt Cancer Center. This project was supported in part by the Research Service of the Veterans' Administration and The Aloe Institute.

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