Chromic-P32 phosphate treatment of implanted pancreatic carcinoma: mechanism involved

Abstract

Aim: To study the effects of chromic-P32 phosphate (32P colloids) interstitial administration in Pc-3 implanted pancreatic carcinoma, and investigate its anticancer mechanism.

Methods: Ninety-eight tumor bearing nude mice were killed at different time points after the injection of 32P colloids to the tumor core with observed radioactivity. The light microscopy, transmission electron microscopy (TEM) and immuno-histochemistry and flow cytometry were used to study the rates of tumor cell necrosis, proliferating cell nuclear antigen index, the micro vessel density (MVD). The changes of the biological response to the lymphatic transported 32P colloids in the inguinal lymph node (ILN) were dynamically observed, and the percentage of tumor cell apoptosis, and Apo2.7, caspase-3, Bcl-2, Bax-related gene expression were observed too.

Results: The half-life of effective medication is 13 d after injection of 32P colloids to the tumor stroma, in 1-6 groups, the tumor cell necrosis rates were 20%, 45%, 65%, 70%, 95% and 4%, respectively (F = 4.14-105.36, P<0.01). MVD were 38.5+/-4.0, 28.0+/-2.9, 17.0+/-2.9, 8.8+/-1.5, 5.7+/-2.3 and 65.0+/-5.2 (t = 11.9-26.1, P<0.01), respectively. Under TEM fairly differentiated Pc-3 cells were found. Thirty days after medication, tumors were shrunk and dried with scabs detached, and those in control group increased in size prominently with plenty of hypodermic blood vessels. In all animals the ILN were enlarged but in medicated animals they appeared later and smaller than those in control group. The extent of irradiative injury in ILN was positively correlated to the dosage of medication. Typical tumor cell apoptosis could be found under TEM in animals with intra-tumoral injection of low dosed 32P colloids. The peak of apoptosis occurred in 2.96 MBq group and 24 h after irradiation. In the course of irradiation-induced apoptosis, the value of Bcl-2/Bax was down regulated; Apo2.7 and caspase-3 protein expression were prominently increased dose dependently.

Conclusion: 32P colloids intra-tumor injection having prominent anticancer effectiveness may reveal the ability of promoting cell differentiation. The low dose 32P colloids may induce human pancreatic carcinoma Pc-3 implanted tumor cell apoptosis; Apo2.7, caspase-3, Bcl-2 and Bax protein participated in regulating the process of irradiation induced cell apoptosis.

Figure 1

Effective half-life in ³²P colloids 7.4 and 14.8 MBq injected group averaged 13 d.

Figure 2

Results of immuno-histochemistry assay of CD34 expression in Pc-3. A: Plenty of stained and densely arranged micro-vessels in tumor field (arrow); B: After medication the tumor cells loosely arranged in tumor field with scanty of micro-vessels stained or no micro-vessels (SABC ×400); PCNA expression in Pc-3. C: In control site, tumor cells were densely arranged with poly nuclei stained in deep brown yellow; D: In each of the dosed sites loosely arranged tumor cells merely with few nuclei stained in light brown yellow color (SABC ×400).

Figure 3

TEM shows the changes in Pc-3 tumor cell; A: Tumor cells (arrow) in control group grew vividly with large nuclei and some nucleoli (12×10³×1.2); B: Tumor cells (arrow) were damaged, only the contour of nucleus remained (6×10³); C: A few remaining tumor cells arranged as a glandular lumen (arrow) with small nucleoli (8×10³×1.2); D: At low dose point, well differentiated Pc-3 cell with small nucleoli, plenty of euchromatin, many long micro-villi on cell surface (arrow), glandular lumens interplaced between the cells having the tendency of exocrine formation (15×10³×1.2).

Figure 4

Transmission electron microscopy of ILN A: Metastatic Pc-3 cells (arrow) in lymphatic tissue of control group (4×10³); B: After ³²P colloids irradiation apoptotic cells (arrow) in the connective tissue and endothelium of lymphatic sinuses (2.5×10³); C: Denatured tumor cells (arrow) after medication (5×10³×1.2); D: ILN under light microscope: lymphatic tissue recovered to normal 4 wk after medication (HE ×400).

Figure 5

Results of TEM: A: Cell necrosis, breaking into debris (arrow) (10×10³); B: Early apoptosis (arrow). Chromatin aggregated at periphery (10×10³×1.2); C: Medium stage of apoptosis (arrow) showing nucleus shrunk and broken (10×10³×1.2).