Mechanism of the interaction of plant alkaloid vincristine with DNA and chromatin: spectroscopic study

Abstract

Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA-cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus.

FIG. 1.

Chemical structure of vincristine.

FIG. 2.

Effect of vincristine on fluorescence emission spectra of DNA and chromatin Excitation was at 258 nm and spectra were recorded between 250 and 270 nm. All samples were prepared in 10 mM Tris–HCl (pH 7.4) and the incubation time after the drug addiction was 45 min. 1–6 are: 0, 5, 10, 15, 25, 50 μg/mL of vincristine, respectively, and DNA concentration was 50 μg/mL. The abbreviation (a.u) is for arbitrary units. I_o and I denote fluorescence emission intensity of DNA (▲) and chromatin (■), respectively, in the absence and presence of vincristine. Stern–Volmer plots of fluorescence quenching of DNA and chromatin is also shown. Results are means±SD (n-3).

FIG. 3.

Near ultraviolet (UV) circular dichroism (CD) spectra of DNA and chromatin in 10 mM Tris–HCl at pH 7.4 in the absence and presence of vincristine. All spectra were recorded between 200 to 320 nm at a scan speed of 2 nm/min at 23°C. 1–5 are 0, 10, 25, 50, 100 and 200 μg/mL of vincristine. The results are means of three individual experiments.

FIG. 4.

Scatchard plots of the binding of vincristine to DNA and chromatin carried out in 10 mM Tris–HCl pH 7.4 for 72 h at room temperature. Inset is r values against free drug (C_f). Results are means of three individual experiments.

FIG. 5.

Absorbance and derivative thermal denaturation profiles of DNA and chromatin in the absence (spectrum 1) and presence of 10, 25, 50, 100 and 200 μg/mL of vincristine (spectra 2–6 respectively). Insets show the absorbance changes of Tm profiles. The samples in 10 mM Tris–HCl (pH 7.4) were continuously heated at 1°C/min. Number of experiments was 3. UV absorbance changes of DNA (■) and chromatin (▲) in the absence and presence of vincristine monitored at 260 nm. The results are presented as means±SD (n=4).

FIG. 6.

Binding affinity of vincristine to single and double strand DNA–cellulose. The resins were equilibrated in 10 mM Tris-HCl pH=7.4 containing 50 mM sodium chloride. Various concentrations of vincristine was added to the resins and incubated for an hour. After centrifugation, the absorbance of the supernatants was monitored at 295 nm. *p=0.05, **p=0.05 and n=3.