Crocin effects on human myeloma cells regarding intracellular redox state, DNA fragmentation, and apoptosis or necrosis profile

Abstract

Background: Well-documented studies reported several pharmacological properties for crocin, the active compound of Crocus sativus, such as its antitumor, radical scavenging, antidepressant, and memory-enhancing effects.

Objectives: We aimed to evaluate the possible cytotoxic activity of crocin on B lymphocytes in human myeloma (U266 cell line) after 24- and 48-hour treatment.

Materials and methods: For this purpose, cell viability was determined by the colorimetric MTT assay and cell death pattern was evaluated using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit. ROS (reactive oxygen species) production and DNA fragmentation were assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA) kit and PI staining, respectively.

Results: The highest concentration of crocin significantly decreased ROS production after 48 hours of treatment. However, crocin had no effect on the expression level of HSP (Heat shock protein). Additionally, its administration caused a mild decline in cell viability and a mild increase in the population of DNA fragmented cells as well as apoptosis.

Conclusions: In our study, no prominent effect was seen; therefore, in order to have a better perspective of crocin activity against cancerous cell lines, further studies are highly recommended.

Keywords: Apoptosis; Crocus; DNA Fragmentation; Necrosis.

Figure 1.. Chemical structure of Crocin

Figure 2.. The Viability of U266 Cells Following 24- and 48-Hour Treatment With Crocin as Assessed by MTT Assay

Data are expressed as mean ± SD of five separate experiments (* and ** represent P < 0.0 and P < 0.01, respectively)

Figure 3.. Crocin Effect on the Percentage of DNA Fragmented Cells Following 24- and 48-Hour Treatments

A and B flow cytometry histograms of treated groups and control after 24 and 48 hours, respectively. C Bar chart illustrates data as mean ± SD of three separate experiments (** represents P < 0.01).

Figure 4.. Assessment of ROS Production in U266 Cells Following 24- and 48-hour Treatment With Crocin Examined by flow Cytometry with DCF-DA

The graph shows and compares mean cell florescent for DCF-DA as mean ± SD in 3 separate experiments (** represents P < 0.01).

Figure 5.. Evaluation of Apoptosis or Necrosis of U266 Cells Following 24 and 48-hour Treatment with crocin Examined by Annexin V/PI Test (early Apoptotic cells Were Annexin V [+] and PI [-], Late Apoptotic cells were Annexin V [+] and PI [+], Necrotic Cells Were Annexin V [-] and PI [+] and the Living cells Annexin V [-] and PI [-])

A and B flow cytometry histograms of treated groups and control after 24 and 48 hours, respectively. C and D bar charts show apoptosis and necrosis as mean ± SD of three separate experiments, respectively. (* and **represent P < 0.05 and P < 0.01, respectively).

Figure 6.. Crocin Effect on the Expression of HSP 70 Protein

A, western blots demonstrating specific bands for HSP 90 and β-actin as an internal control. Equal amounts of protein sample (50 μg) obtained from the cell lysate were applied in each lane. Western blot analysis was carried out for four separate experiments; B, data are expressed as mean ± SD and differences between treatment groups and control are analyzed statistically.

Figure 7.. Crocin Effect on the Expression of HSP 90 Protein

A, western blots demonstrating specific bands for HSP 90 and β-actin as an internal control. Equal amounts of protein sample (50 μg) obtained from the cell lysate were applied in each lane. Western blot analysis was carried out for four separate experiments; B, data are expressed as mean ± SD and differences between treatment groups and control are analyzed statistically.