Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation: Potential mechanisms for its chemotherapeutic effects

Mian Xu, Heather S. Floyd, Suzanne M. Greth, Wen Chi L Chang, Kurt Lohman, Radka Stoyanova, Gregory L. Kucera, Tim E. Kute, Mark C. Willingham, Mark Steven Miller

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Perillyl alcohol (POH) is currently being tested in clinical trials as an anticancer agent, though its mechanism of action has not been definitively established. We treated two human lung cancer cell lines, H322 and H838, with POH to determine its antitumor properties. A sulforhodamine B (SRB) cell proliferation assay was used to determine the effects of POH after 1 and 5 days of treatment with 0.25, 0.5, 0.75, 1.0, and 1.5 mM POH. After 1 day of treatment, little difference could be seen between the lowest and highest concentrations of POH. However, after 5 days, both cell lines showed a dose-dependent decrease in cell proliferation that ranged from 15% to 83%. A clonogenic assay confirmed these results - while there was no significant effect of POH after 1 day of exposure, a dose-dependent decrease in colony formation, ranging from 15% to 100%, was seen after 5 days of treatment. Time-lapse video microscopy revealed that apoptotic cells were evident within 24-48 h of treatment with 1.5 mM POH. The appearance of apoptotic cells was preceded by increased caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP) as POH activated caspase-3 activity 3-6-fold. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) confirmed the classical characteristics of apoptosis in POH-treated cells. DNA microarray expression analysis was performed following 8 and 24 (H322) or 8 and 48 (H838) h of treatment with 1.5 mM POH. While a large number of genes were up- or downregulated in the two cell lines at various times after POH treatment, the levels of expression of only eight genes were up- or down-related in both cell lines at both of the time points examined. The significance of these genes as potential mediators of POH action is still uncertain, but the limited number of commonly up- or downregulated genes detected by microarray expression analysis suggests that POH may mediate its effects via posttranscriptional mechanisms. Our results suggest that POH may have potential use as an anticancer drug that stimulates or sensitizes lung tumor cells to apoptosis, and this effect may depend on genetic lesions present in tumor cells.

Original languageEnglish
Pages (from-to)232-246
Number of pages15
JournalToxicology and Applied Pharmacology
Volume195
Issue number2
DOIs
StatePublished - Mar 1 2004
Externally publishedYes

Fingerprint

perilla alcohol
Lung Neoplasms
Cells
Cell Proliferation
Cell Line
Genes
Cell proliferation
Microarray Analysis
Microarrays
lissamine rhodamine B
Caspase 3
Tumors
Assays
Down-Regulation

Keywords

  • 4′,6-diamidino-2- phenylindole
  • Apoptosis
  • DAPI
  • Ethanol
  • EtOH
  • LOH
  • Loss of heterozygosity
  • Lung cancer
  • Microarray
  • Monoterpene
  • Non-small cell lung cancer
  • NSCLC
  • PARP
  • Perillyl alcohol
  • POH
  • Poly (ADP-ribose) polymerase
  • Relative light unit
  • RLU
  • SRB

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation : Potential mechanisms for its chemotherapeutic effects. / Xu, Mian; Floyd, Heather S.; Greth, Suzanne M.; Chang, Wen Chi L; Lohman, Kurt; Stoyanova, Radka; Kucera, Gregory L.; Kute, Tim E.; Willingham, Mark C.; Miller, Mark Steven.

In: Toxicology and Applied Pharmacology, Vol. 195, No. 2, 01.03.2004, p. 232-246.

Research output: Contribution to journalArticle

Xu, Mian ; Floyd, Heather S. ; Greth, Suzanne M. ; Chang, Wen Chi L ; Lohman, Kurt ; Stoyanova, Radka ; Kucera, Gregory L. ; Kute, Tim E. ; Willingham, Mark C. ; Miller, Mark Steven. / Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation : Potential mechanisms for its chemotherapeutic effects. In: Toxicology and Applied Pharmacology. 2004 ; Vol. 195, No. 2. pp. 232-246.
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T2 - Potential mechanisms for its chemotherapeutic effects

AU - Xu, Mian

AU - Floyd, Heather S.

AU - Greth, Suzanne M.

AU - Chang, Wen Chi L

AU - Lohman, Kurt

AU - Stoyanova, Radka

AU - Kucera, Gregory L.

AU - Kute, Tim E.

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AU - Miller, Mark Steven

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N2 - Perillyl alcohol (POH) is currently being tested in clinical trials as an anticancer agent, though its mechanism of action has not been definitively established. We treated two human lung cancer cell lines, H322 and H838, with POH to determine its antitumor properties. A sulforhodamine B (SRB) cell proliferation assay was used to determine the effects of POH after 1 and 5 days of treatment with 0.25, 0.5, 0.75, 1.0, and 1.5 mM POH. After 1 day of treatment, little difference could be seen between the lowest and highest concentrations of POH. However, after 5 days, both cell lines showed a dose-dependent decrease in cell proliferation that ranged from 15% to 83%. A clonogenic assay confirmed these results - while there was no significant effect of POH after 1 day of exposure, a dose-dependent decrease in colony formation, ranging from 15% to 100%, was seen after 5 days of treatment. Time-lapse video microscopy revealed that apoptotic cells were evident within 24-48 h of treatment with 1.5 mM POH. The appearance of apoptotic cells was preceded by increased caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP) as POH activated caspase-3 activity 3-6-fold. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) confirmed the classical characteristics of apoptosis in POH-treated cells. DNA microarray expression analysis was performed following 8 and 24 (H322) or 8 and 48 (H838) h of treatment with 1.5 mM POH. While a large number of genes were up- or downregulated in the two cell lines at various times after POH treatment, the levels of expression of only eight genes were up- or down-related in both cell lines at both of the time points examined. The significance of these genes as potential mediators of POH action is still uncertain, but the limited number of commonly up- or downregulated genes detected by microarray expression analysis suggests that POH may mediate its effects via posttranscriptional mechanisms. Our results suggest that POH may have potential use as an anticancer drug that stimulates or sensitizes lung tumor cells to apoptosis, and this effect may depend on genetic lesions present in tumor cells.

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