Intraperitoneal photodynamic therapy for an ovarian cancer ascite model in Fischer 344 rat using hematoporphyrin monomethyl ether

Abstract

With limited treatment options, intraperitoneal spread of ovarian cancer is a common problem leading to high morbidity. Intraperitoneal photodynamic therapy combined with debulking surgery to treat residual disease is an alternative choice for clinicians. Hematoporphyrin monomethyl ether (HMME) is a promising second-generation photosensitizer developed in China. Our study was designed to investigate the phototoxicity of HMME on ovarian cancer. NuTu-19, a cell line derived from adenocarcinoma of Fischer 344 rat, and its allogeneic graft ascites tumor model was used in this study. HMME was confirmed to be localized in cytolysosome, and HMME-based photosensitization induced direct necrosis as well as mitochondria damage. The photocytotoxicity of HMME was both light- and drug dose-dependent and no significant dark cytotoxicity was observed in NuTu-19 cells. With the ascite tumor-bearing Fischer 344 rat model, HMME-based intraperitoneal photodynamic therapy was proved to be useful in improving the prognosis of ovarian cancer. Thus, this study provides evidence that HMME-based photodynamic therapy is an effective adjuvant therapy for ovarian cancer.

Figure 1

Subcellular localization of hematoporphyrin monomethyl ether (HMME) in NuTu‐19 cells derived from adenocarcinoma of Fischer 344 rat. Fluorescence images of the cells double‐stained with HMME and mitochondria fluorescent probe (Rhodamine‐123; Sigma) or cytolysosome fluorescent probe (Lucifer Yellow; Sigma) are shown. Cells were observed at various time intervals (0.5, 1, 2, 3, 6, and 12 h) following coincubation of HMME and Rhodamine‐123 or Lucifer Yellow. (a) HMME + Rhodamine‐123; (b) HMME + Lucifer Yellow; (c) HMME alone; (d) Rhodamine‐123 alone; (e) Lucifer Yellow alone. All photos were taken 3 h after incubation.

Figure 2

Photocytotoxity of hematoporphyrin monomethyl ether (HMME) to NuTu‐19 cells derived from adenocarcinoma of Fischer 344 rat. Shown are the cell survival rates at 24 h after photodynamic treatment with different concentrations of HMME (0–20 µg/mL) and different light doses (0–6 J/cm²).

Figure 3

Mitochondria damage in NuTu‐19 cells, derived from adenocarcinoma of Fischer 344 rat, following hematoporphyrin monomethyl ether photodynamic treatment. (a) Orange fluorescent spots in control cells indicate the intact mitochondria. (b) Mitochondria fluorescent spots disappeared completely and there were only diffused weaker green fluorescent spots in the cytoplasm, indicating the mitochondria damage.

Figure 4

Cell death mode induced by hematoporphyrin monomethyl ether‐based photodynamic treatment (PDT). Flow cytometry analysis of NuTu‐19 cancer cells, derived from adenocarcinoma of Fischer 344 rat. with Annexin V/propidium iodide (PI) double staining after PDT. (a) Controls; (b) 4 h after PDT; (c) 8 h after PDT. UL (upper left quadrant): Annexin V(–)∖PI(+), cell fragment; UR (upper right quadrant): Annexin V(+)∖PI(+), necrosis cells; LL (lower left quadrant): Annexin V(–)∖PI(–), survival cells; LR (lower right quadrant): Annexin V(+)∖PI(–), apoptotic cells.

Figure 5

Kaplan–Meier survival curves for tumor‐bearing Fischer 344 rats. Treatment groups (n = 9) included animals that received cytoreductive surgery and intraperitoneal photodynamic treatment (hematoporphyrin monomethyl ether 10 mg/kg, laser 50 J/cm²). Control groups included animals that received cytoreductive surgery alone (n = 8) or cytoreductive surgery followed by laser illumination (50 J/cm²) without photosensitizer (n = 9).