Paclitaxel impairs adipose stem cell proliferation and differentiation

Abstract

Background: Cancer patients with chemotherapy-induced immunosuppression have poor surgical site wound healing. Prior literature supports the use of human adipose-derived stem cell (hASC) lipoinjection to improve wound healing. It has been established that multipotent hASCs facilitate neovascularization, accelerate epithelialization, and quicken wound closure in animal models. Although hASC wound therapy may benefit surgical cancer patients, the chemotherapeutic effects on hASCs are unknown. We hypothesized that paclitaxel, a chemotherapeutic agent, impairs hASC growth, multipotency, and induces apoptosis.

Methods: hASCs were isolated and harvested from consented, chemotherapy and radiation naive patients. Growth curves, MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide), and EdU (5-ethynyl-2-deoxyguridine) assays measured cytotoxicity and proliferation. Oil Red O stain, Alizarin Red stain, matrigel tube formation assay, and quantitative polymerase chain reaction analyzed hASC differentiation. Annexin V assay measured apoptosis. Immunostaining and Western blot determined tumor necrosis factor α (TNF-α) expression.

Results: hASCs were selectively more sensitive to paclitaxel (0.01-30 μM) than fibroblasts (P < 0.05). After 12 d, paclitaxel caused hASC growth arrest, whereas control hASCs proliferated (P = 0.006). Paclitaxel caused an 80.6% reduction in new DNA synthesis (P < 0.001). Paclitaxel severely inhibited endothelial differentiation and capillary-like tube formation. Differentiation markers, lipoprotein lipase (adipogenic), alkaline phosphatase (osteogenic), CD31, and van Willebrand factor (endothelial), were significantly decreased (all P < 0.05) confirming paclitaxel impaired differentiation. Paclitaxel was also found to induce apoptosis and TNF-α was upregulated in paclitaxel-treated hASCs (P < 0.001).

Conclusions: Paclitaxel is more cytotoxic to hASCs than fibroblasts. Paclitaxel inhibits hASC proliferation, differentiation, and induces apoptosis, possibly through the TNF-α pathway. Paclitaxel's severe inhibition of endothelial differentiation indicates neovascularization disruption, possibly causing poor wound healing in cancer patients receiving chemotherapy.

Keywords: Cancer therapy; Chronic wounds; Human adipose–derived stem cells; Neovascularization; Paclitaxel; Wound healing.

Figure 1

Establishing human adipose derived stem cell lines (hASC). Starting with patient lipoaspiration, followed by digestion with collagenase and cetrifugation to create the stem cell pellet. After hASC incubation, culture in different differentiation mediums results in adipocyte, osteocyte, and endothelial cell development and the possibility for clinical application such as improving wound healing with hASCs.

Figure 2

The molecular form of Paclitaxel. Paclitaxel is a chemotherapeutic agent often used in breast, ovarian, and lung cancer. It disrupts microtubule organization causing mitotic arrest and induces cancer cell death via apoptosis.

Figure 3

The effect of Paclitaxel on hASC viability. A. Cytotoxicity of Paclitaxel in hASCs and fibroblasts after 1 day of treatment. MTT Assay revealed hASCs were selectively more cytotoxic to Paclitaxel than fibroblasts. Both 0.01μM and 1μM doses of Paclitaxel were significantly more cytotoxic to hASCs than fibroblasts (p=0.02 and p=0.008). B. Cytotoxicity of Paclitaxel in hASCs and fibroblasts after 3 days of treatment. MTT Assay revealed a similar trend to the 1-day treatment; hASCs were selectively more cytotoxic to Paclitaxel than fibroblasts at the following doses: (0.1μM: p=0.025, 1μM: p=0.011, 3μM: p=0.01, and 30μM: p=0.045).

Figure 4

The effect of Paclitaxel (PTX) on hASC growth. A. A cell growth curve of hASCs with and without Paclitaxel treatment. The number of control hASCs increased steadily while Paclitaxel treated hASCs experienced growth arrest after 12 days (p=0.006). B. Images after EdU Assay measured DNA incorporation in hASCs with and without Paclitaxel treatment. C. Analyzed DNA incorporation in hASCs with and without Paclitaxel shown by bar graph. Paclitaxel treated hASCs had significantly less proliferation and DNA synthesis than control hASCs as measured by EdU DNA incorporation (p <0.001).

Figure 5

The effect of Paclitaxel (PTX) on hASCs adipogenic and osteogenic differentiation. A. Adipogenic differentiation. Oil Red O stain showed there were less stained lipid droplets in the adipogenic-induced hASCs treated with Paclitaxel as compared to control hASCs. B. Osteogenic differentiation. Alazarin red stain illustrated the calcium deposits seen in osteogenic-induced hASCs treated with and without Paclitaxel. C. Adipogenic markers, LPL and PPAR-γ, revealed reduced expression in Paclitaxel treated hASCs. (p=0.043, p=0.345). D. Osteogenic markers, alkaline phosphatase and osteopontin, revealed reduced expression in Paclitaxel treated hASCs. (p=0.043, p=0.345).

Figure 6

The effect of Paclitaxel (PTX) on hASC endothelial differentiation. A. Bright field cell images of control hASCs and Paclitaxel treated hASCs in 6-well plates and Matrigel coated 24-well plates. Light microscopy showed Paclitaxel treatment induced cell morphology changes and less tube formation. B. The expression of endothelial differentiation markers CD31, vWF, and eNOS, measured by Real-time PCR. Two of the three endothelial markers, CD31 and vWF, revealed significantly reduced expression in Paclitaxel treated hASCs (p=0.016, p=0.031, p=0.500).

Figure 7

The Annexin V apoptosis detection kit using flow cytometry determined an increasing number of Paclitaxel (PTX) treated hASCs entered early apoptosis as compared to untreated control hASCs. A. 15.8% of control cells in the early apoptotic phase. B. After 24 hours of Paclitaxel treatment, 18.8% of hASCs were in early apoptosis. C. After 3 days of Paclitaxel treatment, 30.8% of hASCs were in early apoptosis. D. When 1 dose of Paclitaxel was given, followed by medium changes without Paclitaxel for 7 days, 51.8% of hASCs were in early apoptosis. E. After 7 days of continual treatment with Paclitaxel, 64.7% of hASCs were in early apoptosis.

Figure 8

The localization and expression of TNF-α in control and Paclitaxel (PTX) treated hASCs. A. Immunostaining revealed Paclitaxel treated hASCs had an increased amount of TNF-α as compared to control hASCs. B. A representative western blot image for TNF and β-actin in control and Paclitaxel treated hASCs. C. Analyzed Western blot data for 4 hASC lines, the expression of TNF-α protein was normalized by β-actin (p<0.001).

Figure 9

Human adipose derived stem cell (hASC) function with and without Paclitaxel. A. The natural course of hASC differentiation. B. The inhibiting effect of Paclitaxel on hASC differentiation and Paclitaxel’s induction of apoptosis, possibly through TNF-α’s upregulation.