Medicine for HIV AIDS, hepatitis c virus infection, cancer treatment

   aids and cancer treatment

 aids and cancer treatment                                                                                Biochemical Pharmacology

 ELSEVIER                                                                                               Biochemical Phannaco1ogy 7194 (2002) 1-7

The alkaloid sanguinarine is effective against multi drug resistance in human cervical cells via bimodal cell death

Zhihu Ding (a), Shou-Ching Tang (a,b), Priya Weerasinghe (a), Xiaolong Yang (a), Alan Pater(a,*), Andrejs Liepins (a)

a -"Division of Basic Sciences, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Philip Drive, St. Johns, NF, Canada AiB 3V6
b - Newfoundland Cancer Treatment and Research Foundation, DI: H. Bliss Murphy Cancer Cente/;
300 Prince Philip Drive, St. Johns, NF, Canada AiB 3V6
Received 1 June 2001; accepted 12 October 2001


  Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 uM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by western blot analysis. However, 8.48 and 16.96 uM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status. C 2002 Published by Elsevier Science Inc.
Keywords: Alkaloid sanguinarine; Multidrug-resistant cervical cells; Apoptosis; Oncosis;Caspase-3

1. Introduction

   Sanguinarine (Scheme 1) is derived from the plant Sanguinaria canadensis [1]. Its principal pharmacologic use to date is in dental products based on its antibacterial, antifungal, and anti-inflammatory activities, which reduce gingival inflammation and supragingival plaque formation [2-4]. Sanguinarine also has been reported to have antiviral and tumor-targeting activity [5-7].
  Molecular biological studies indicate that sanguinarine has multiple cellular targets [8]. For example, it can interact with and intercalate DNA [9,10], inhibit micro- tubule assembly [11], affect membrane permeability [12,13], and inhibit a wide variety of enzymes, including Na+/K+ ATPase [14]. Most interestingly, it also is a potent inhibitor of protein kinases [15] and NF-KB [16], which are involved in signal transduction pathways leading to cell proliferation and/or cell death [7].
  Cell death is important for normal homeostasis, cell proliferation, and differentiation. The importance of cell death is demonstrated by the observation that dysregulation of cell death can lead to cancer, developmental abnormalities, and autoimmune disorders [17-19]. Cells undergoing PCD (or apoptosis) are characterized by morphologic changes, including cellular shrinkage, blebbing, and nuclear DNA condensation with or without fragmentation [20-24]. However, it is stated that apoptosis is rarely observed in vivo and may not be the sole mechanism of cell death [25]. The discovery of intact novel forms of cell death pathways induced by potential anticancer agents may have an important bearing in overcoming chemoresistance.
  Of all neoplasms found in females worldwide, cervical cancer has the third highest incidence and is fourth on the list of the leading causes of death by cancer [26,27]. The available drugs most commonly used for treating cervical malignancies are impeded by frequent progression to chemotherapy resistance. Sanguinarine may be effective against MDR, since the related Sanguinaria canadensis-derived alkaloid, chelerythrine, has been shown to be cytotoxic to cancer cells and MDR cells [28]. In this study, we used our recently established in vitro cervical cancer model system for MDR [29] to investigate whether sanguinarine is effective against MDR in human cervical cells, and to understand the cellular and molecular mechanisms by which it may induce cell death.

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2. Materials and methods

2.1. Cell culture, cell viability assays, and clonogenic survival assays

  Most cell culture protocols, the HPV type 16-immortalized human endocervical cell line (HEN-16-2), the CSC- transformed HEN-16-2 cell line (HEN-16-2T), and the MDR HEN-16-2 cell line (HEN-16-2/CDDP) have been described previously [29-31]. Cells were cultured in keratinocyte growth medium (KGM). HeLa cervical carcinoma, CEM- VLB leukemia, CEM- T4 leukemia, K562 erythroleukemia, and 1M1 pre-B cell lymphoblastic cells were obtained from the American Type Culture Collection. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). CEM- VLB leukemia, CEM- T4 leukemia, K562 erythroleukemia, and 1M1 pre-B cell lymphoblastic cells were cultured in RPMI-1640 medium supplemented with 10% FBS. All experiments were performed in triplicate.
  Sanguinarine chloride (Sigma) was dissolved in H2O as a 2.72 M stock solution, aliquots of which were serially diluted with KGM and used when needed to prepare fresh working solutions.
  To examine the effect of sanguinarine on cell viability, 5 x 104 cells/well were seeded in 12-well plates, incubated for 4 or 48 hr, treated with various concentrations of sanguinarine, and then assayed for trypan blue exclusion and propidium iodide exclusion under light microscopy, as described previously [32,33]. A hemocytometer was used to count the cells.
  Clonogenic survival assays were performed to examine the combined survival and proliferative potential of sanguinarine-treated cervical cells, as previously described [29,34]. Briefly, 103 cells were seeded int060-mm plates, incubated with 0-16.96 uM sanguinarine for 24 hr, washed twice with phosphate-buffered saline, and incubated without sanguinarine for 10-14 days. The cells were stained with 2% (w/v) crystal violet in methanol, and colonies of 50 or more cells were counted using a hemocytometer.

2.2. Cell morphology analysis

  To examine the effect of sanguinarine on cell morphology under light microscopy, 5 x 103 cells/chamber were seeded in 8-chamber slides (Nalge Nunc International), incubated for 24 hr, and treated with 0-16.96 J.lM sanguinarine for 4 hr.
  To examine the cell ultrastructural effect of sanguinarine under scanning electron microscopy (SEM), 5 x 104 cells/ well were seeded in 12-well plates containing acid-cleaned coverslips (Lux Scientific Corp.), incubated for 24 hr for attachment to coverslips, treated with 0-16.96 uM sanguinarine for 4 hr, fixed in Karnovsky fixative containing 2.5% (v/v) glutaraldehyde (J.B. EM Services) in 0.1 M sodium cacodylate buffer, and then dehydrated in a 25, 50, 75, and 100% (v/v) ethanol series followed by Freon 113 substitution. All samples were dried simultaneously, sputter-coated with gold, and examined under a Hitachi S-570 scanning electron microscope, as previously described [35].


2.3. Western blot analysis

  Western blot analysis of the effect of sanguinarine on caspase-3 activation and PARP cleavage was performed as described previously [36]. Briefly, 10 J.1g of protein was resolved by 10% (w/v) SDS-PAGE and transferred to Hybond enhanced chemiluminescence (ECL) nitrocellu- lose membrane under semidry conditions. Irnrnunodetec- tion was performed using the ECL system (Amersham Pharmacia Biotech). Procaspase-3 and caspase-3 were probed using anti-caspase-3 monoclonal antibody (mAb) (Santa Cruz Biotechnology). Full-length and cleaved frag- ments ofPARP were probed usinganti-PARP mAb (Phar- Mingen).

3. Results

3.1. Evasion of MDR of human cervical HEN-16-2/ CDDP cells by sanguinarine

Table I
Bimodal cell death characteristics induced by sanguinarine and Ukrain in cervical cells and leukemia cells

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HEN-16-2a                    PCD/+/+                    BCD/-/-                            PCD/+/+                        BCD/-/-
HEN-16-2/CDDpb          PCD/+/+                    BCD/-/-                            PCD/+/+                        BCD/-/-

HEN-16-2T"                 PCD/+/+                    BCD/-/-                            PCD/+/+                        BCD/-/-
HeLad                          PCD/+/+                    BCD/-/-                            PCD/+/+                        BCD/-/-
CEM- T4.                    PCD/NA/NA               BCD/NA/NA                    NA/NA/NA                     NA/NA/NA

CEM-VLBf                   PCD/NA/NA               BCD/NA/NA                    NA/NA/NA                     NA/NA/NA
JMlg                           PCD/NA/NA               BCD/NA/NA                    PCD/NA/NA                   BCD/NA/NA
K562h                         PCD/NA/NA               BCD/NA/NA                    PCD/NA/NA                   BCD/NA/NA

   Cells were treated with a dilution series of sanguinarine or Ukrain for 4 hr and morphologic changes were observed by microscopy. For examining caspase-3 activation and PARP cleavage, cell lysates were subjected to western blotting using anti-caspase-3 mAb and anti-PARP mAb. PCD programmed cell death or apoptosis; BCD, blister cell death or oncosis; NA, not available.
   a Human endocervical (HEN) immortalized with HPVI6.
   b HEN-l 6-2 transformed by cisplatin and MDR.
   c HEN-16-2 transformed by cigarette smoke condensate.
   d Endocervical carcinoma.
   e Leukemia P-gp-negative.
   f Leukemia P-gp-positive.
   g Pre-B cell lymphoblastic cell line, Bcl-2 high level.
   h Erythroleukemia cell line, Bcl-210w level.

    We examined the chemotherapeutic potential of sanguinarine for MDR cervical cancer cells in a human cervical in vitro system, which is composed of MDR HEN-I6-2/ CDDP cells and their drug-sensitive parental HEN-I6-2 cells [29]. Cell viability, measured by the trypan blue exclusion assay, was similar in both types of cells treated with 0, 0.13, 0.26, 0.53, 1.06,2.12, and 4.24 uM sanguinarine for 4 or 48 hr (Table 1; Fig. 1). The propidium iodide exclusion assay also showed no significant difference in cell viability between MDR HEN-16-2/CDDP cells and their parental HEN-16-2 cells after treatment with these concentrations of sanguinarine for 4 or 48 hr (data not shown). Treating the MDR HEN-16-2/CDDP and HEN- 16-2 cells with 0-1.06 uM sanguinarine produced no significant increase in cell viability; however, 2.12 uM (Fig. 2) and 4.24 uM sanguinarine treatment caused the death of most of the cells (data not shown). Treating HEN-16-2/ CDDP and HEN-16-2 cells with 8.48 and 16.96 uM sanguinarine resulted in 100% cell death within 48 hr (data not shown). Clonogenic survival assays (also called colony-forming assays) revealed no significant difference in clonogenic survival between HEN-16-2/CDDP and HEN-16- 2 cells (data not shown), showing an equally effective potential of sanguinarine in this assay to kill cells and inhibit their growth.

aids and cancer treatment

Fig. I. Concentration-dependent effect of sanguinarine on HEN-16-2 and HEN-16-2/CDDP cell viability. Cells were incubated with 0, 0.13, 0.26, 0.53, 1.06, and 2.12 uM sanguinarine for 48 hr. Cell viability represents the percentage of treated compared with untreated cells that excluded trypan blue dye. The results represent the means +/- SD of three independent experiments.

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Fig. 2. Concentration-dependent bimodal effect of sanguinarine on the morphology of MDR HEN-16-2/CDDP cells. The panels represent untreated control cells under light microscopy (A) and scanning electron microscopy (SEM) (B); 4 hr, 2.12 uM sanguinarine-treated cells under light microscopy (C) and SEM (D); and 4 hr, 8.48 uM sanguinarine-treated cells under light microscopy (E) and SEM (F).

3.2. Induction of concentration-dependent apoptosis and oncosis in MDR HEN-I6-2/CDDP and drug-sensitive HEN-I6-2 cervical cells by sanguinarine

    To evaluate the concentration-dependent effect of sanguinarine on cell death morphology, cells were treated with different concentrations of sanguinarine for 4 hr and observed microscopically. Both cell lines treated with 0- 1.06 uM sanguinarine were observed to have normal cell morphology, similar to the morphology of untreated MDR cells (Table 1; Fig. 2A and B). Cell plasma membrane blebbing, a characteristic of PCD or apoptosis, was observed in cells treated for 4 hr with 2.12 uM sanguinarine (Fig. 2C and D) and 4.24 uM sanguinarine (Table I). However, most cells exhibited single and rare double cell surface blisters after sanguinarine treatment for 4 hr with 8.48 uM (Fig. 2E and F) and 16.96 uM (Table I). Similar bimodal apoptosis and BCD (or oncosis) were observed at the same respective sanguinarine concentrations and time in CSC-transformed HEN-16-2T, HeLacells, MDR CEM-VLB leukemia, drug-sensitive CEM-T4 leukemia, K562 erythroleukemia, and JM1 pre-B cell lymphoblastic cells (Table I).


32kDa-1               ______________ 0.5 hr __________      __________4 hr _______________

17kDa-             aids and cancer treatment


(B)                                                                                                                                         PARP

116 kDa-                                                                                                                                 -+ Precursor

85 kDa-                 aids and cancer treatment    -+ Cleaved product

Fig. 3. Concentration- and time-dependent caspase-3 activation and PARP cleavage in sanguinarine-treated MDR HEN-16-2/CDDP cells. Western blot analysis is shown for caspase-3 (A) and PARP (B) using 10 ug protein/lane from cells treated for 0.5 or 4 hr with sanguinarine at 0 uM (lanes I and 7), 1.06 uM (lanes 2 and 8), 2.12 uM (lanes 3 and 9), 4.24 uM (lanes 4 and 10), 8.48 uM (lanes 5 and II), and 16.96 uM (lanes 6 and 12).

3.3. Induction of caspase-3 activation in apoptosis but
not oncotic cell death in both MDR HEN- I 6-2/CDDP and drug-sensitive HEN-16-2 cells by sanguinarine

   To study the molecular mechanism by which sanguinarine induces cell morphologic changes, sanguinarine-treated cells were examined for the proteolytic activation of caspase-3, a downstream effector in apoptosis pathways. Sanguinarine induced time- and concentration-dependent activation of caspase-3 in MDR HEN-16-2/CDDP cells, as observed by western blotting (Fig. 3A). Treatment for 0.5 hr with 0-16.96 uM sanguinarine did not cause detectable proteolytic activation of caspase-3; a 4-hr treatment with 2.12 and 4.24 uM sanguinarine, but no other concentration from 0 to 16.96 uM, induced cleavage of procaspase-3 to the activated 17 kDa caspase-3 fragment. These results are consistent with a previous demonstration that apoptosis requires caspase-3 activation [37].
  PARP is a critical cellular substrate for proteolysis by activated caspase-3 [38]. Therefore, we also studied whether the activation of caspase-3 by sanguinarine may lead to increased cleavage of PARP. In a time- and concentration-dependent analysis of PARP cleavage that parallels the one for caspase-3 activation, cleaved PARP fragments were found at only 4 hr in 2.12 and 4.24 uM sanguinarine-treated MDR cells (Fig. 3B) and drug-sensitive cells (Table 1). For several other cervical cell lines, including HEN-16-2T, similar sanguinarine concentration- and time-dependent caspase-3 and PARP results were observed indicating apoptosis; both results were absent in BCD/oncosis (Table 1). Overall, these results suggest that sanguinarine may be equally effective against MDR and drug-sensitive human cervical cells, and act despite MDR through bimodal apoptosis and BCD/oncosis pathways having mechanisms that involve differential morphologies and caspase-3 activation status.

4. Discussion

  We established the in vitro MDR cervical cell system used in this report by treating HPV 16-immortalized human endocervical HEN-16-2 cells with cisplatin [29]. Cell viability was significantly higher in the MDR HEN-16- 2/CDDP cells than in the parental cells after treatment with cisplatin, actinomycin D, doxorubicin, etoposide, paclitaxel, 5-fluorouracil, staurosporine, heat shock, or UV radiation [29,39]. However, this study found no significant difference in the effect of sanguinarine on cell viability or clonogenic survival between the MDR HEN-16-2/CDDP cells and their parental drug-sensitive HEN-16-2 cells. Similarly, there was no significant difference in cell death induced by sanguinarine between CEM- VLB leukemia cells in which P-glycoprotein (P-gp) mediates MDR and their wild-type drug-sensitive counterpart CEM- T4 cells, which are P-gp-negative (Table I). Importantly, sanguinarine has been found to be selectively less toxic to normal cells [7]. Thus, sanguinarine may be regarded as a potential therapeutic agent even for MDR of certain types of transformed cells, which are represented by HEN-16-2/CDDP cells [29].

    Proteolytic activation of effector caspases, especially caspase-3, is one of the key events in apoptosis [38,40]. The results presented here show that sanguinarine induced both apoptosis and BCD/oncosis in cervical MDR HEN- 16-2/CDDP cells and drug-sensitive HEN-16-2 cells. Lower concentrations of sanguinarine induced apoptosis, displayed by cell surface blebbing (Fig. 2C and D) and caspase-3 activation, the latter confirmed by induction of proteolytic cleavage of the caspase-3 substrate PARP (Fig. 3). Higher concentrations of sanguinarine induced cell death characterized by blistering, an oncotic late cell death observed previously [41], and the absence of caspase-3 activation (Figs. 2E and F and 3).
   Bimodal cell death was also found to be induced by sanguinarine in K562 erythroleukemia cells [42], JMl pre-B lymphoblastic cells [42], MDR CEM- VLB leukemia, and their wild-type counterpart CEM-T4 cells (Table 1). Ukrain, an alkaloid derived from the same plant family as sanguinarine, has been reported to also induce apoptotic and blister forms during K562leukemia cell death [21], and in MDR HEN-16-2/CDDP and drug-sensitive HEN-16-2 cells (Table 1). Electronic transmission microscopy of K562 cells showed that sanguinarine-induced apoptosis produced classic morphologic changes, including the formation of apoptotic bodies containing organelles and chromatin condensation [42], whereas sanguinarine-induced oncosis produced blisters that were devoid of organelles and displayed patchy chromatin condensation [42].
   BCD/oncosis is a form of cell death that is distinct from apoptosis [43], whereas necrosis refers to the intracellular degradative reactions occurring after cell death by any mechanism, including apoptosis [44]. Oncosis has been documented in many studies [45-50]. The molecular and biochemical mechanisms underlying oncosis are still unclear. Oncosis was believed to result from a failure of plasma membrane ionic pumps and decreased levels of cellular ATP [51]. However, cell surface proteins, including phospholipase A2 and Porimin, have been documented to be involved in the process of cell membrane injury and membrane structural changes [49,50,52]. Sanguinarine- induced cell death pathways may be initiated that, if not blocked, lead to caspase-3 activation, cleavage of PARP and other caspase-3 substrates, and consequent apoptotic cell death. If these pathways are blocked, then other downstream or parallel steps of a pathway may lead to caspase-independent oncosis [53-58]. Future studies on the sanguinarine-activated cellular factors involved in cell death pathways may provide a greater understanding of the bimodal cell death pathways.
  In summary, the data in this report indicate that sanguinarine induces concentration-dependent apoptosis with caspase-3 activation and BCD/oncosis without caspase-3 activation. The ability of this drug to induce bimodal cell death modes at comparable efficiencies in MDR and drug- sensitive human cervical and leukemia cells indicates that sanguinarine was effective against MDR in this in vitro system, and that there may be two sanguinarine-induced cell death mechanisms.


  We thank Mr. G. Chemenko, Ms. Y. Hao, and Ms. L. Lee for excellent technical assistance. The work was supported by a National Cancer Institute of Canada grant (2734 to A.P.) with funds from the Canadian Cancer Society; Medical Research Council of Canada grants (MT -9782 and MT- 10140 to A.P. and MT-13178 to A.L.), and a Canadian Institutes of Health Research (CIHR) grant (ROP-40859 to A.P.).


  [I] Shamma M, Guinaudeau H. Aporphinoid alkaloids. Nat Prod Rep 319 1986;3:345-51.
  [2] Kuftinec MM, Mueller-Joseph LJ, Kopczyk RA. Sanguinaria tooth- 321 paste and oral rinse regimen clinical efficacy in short- and long-tenD 322 trials. J Can Dent Assoc 1990;56:31-3. 323
  [3] Laster LL, Lobene RR. New perspectives on Sanguinaria clinicals: 324 individual toothpaste and oral rinse testing. J Can Dent Assoc 1990; 325 56:19-30. 326
 [4] Godowski KC, Wolff ED, Thompson DM, Housley CJ, Polson AM, 327 Dunn RL, Duke SP, Stoller NH, Southard GL. Whole mouth 328 microbiota effects following subgingival delivery of sanguinarium. J 329 PeriodontoI1995;66:870-7. 330
 [5] Colombo ML, Bosisio E. Pharmacological activities of Chelidonium 331 majus L. (Papaveraceae). Pharmacol Res 1996;33:127-34. 332
 [6] Faddeeva MD, Beliaeva TN. Sanguinarine and ellipticine cytotoxic 333 alkaloids isolated from well-known antitumor plants. Intracellular 334 targets of their action. Tsitologiia 1997;39:181-208. 335
 [7] Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H. 336 Differential antiproliferative and apoptotic response of sanguinarine 337 for cancer cells versus normal cells. Clin Cancer Res 2000;6:1524-8. 338
 [8] Walterova D, Ulrichova J, Valka I, Vicar J, Vavreckova C, Taborska 339 E, Harjrader RJ, Meyer DL, Cerna H, Simanek V. Benzo[c]phenan-
     thridine alkaloids sanguinarine and chelerythrine: biological activ- 341 ities and dental care applications. Acta Univ Palacki Olomuc Fac 342 Med 1995;139:7-16. 343
 [9] Nandi R, Maiti M. Binding of sanguinarine to deoxyribonucleic acids 344 of differing base composition. Biochem PharmacoI1985;34:321-4. 345
[10] Saran A, Srivastava S, Coutinho E, Maiti M. IH NMR investigation 346 of the interaction of berberine and sanguinarine with DNA. Indian J 347 Biochem Biophys 1995;32:74-7. 348
[II] Wolff J, Knipling L. Antimicrotubule properties of benzophenan- 349 thridine alkaloids. Biochemistry 1993;32:13334-9. 350
[12] Babich H, Zuckerbraun HL, Barber IB, Babich SB, Borenfreund E. 351 Cytotoxicity of sanguinarine chloride to cultured human cells from 352 oral tissue. Pharmacol ToxicoI1996;78:397-403. 353
[13] Schmeller T, Latz-Bruning B, Wink M. Biochemical activities of 354 berberine, palmatine and sanguinarine mediating chemical defence 355 against microorganisms and herbivores. Phytochemistry 1997;44:257- 356 66. 357
[14] Das M, Khanna SK. Clinicoepidemiological, toxicological, and safety 358 evaluation studies on argemone oil. Crit Rev Toxicol 1997;27:273- 359 97. 360
[15] Wang BH, Lu ZX, Polya GM. Inhibition of eukaryote protein kinases 361 by isoquinoline and oxazine alkaloids. Planta Med 1997;63:494-8. 362
[16] Chaturvedi MM, Kumar A, Damay BG, Chainy GB, Agarwal S, 363 Aggarwal BB. Sanguinarine (pseudochelerythrine) is a potent inhibitor 364 of NF-KB         activation, IKB-(X phosphorylation, and degradation. J Bioi Chem 1997;272:30129-34.
 [17] OITenius S. Apoptosis: molecular mechanisms and implications for human disease. J Intern Med 1995;237:529-36.
 [18] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995 ;267: 1456-62.
 [19] White E. Life, death, and the pursuit of apoptosis. Genes Dev 1996;10:1-15.
 [20] Liepins A. Morphological, physiological and biochemical parameters associated with cell injury: a review. Immunopharmacol Immunotox- icoI1989;11:539-58.
 [21] Liepins A, Nowicky JW, Bustamante JO, Lam E. Induction of bimodal programmed cell death in malignant cells by the derivative Ukrain (NSC-631570). Drugs Exp Clin Res 1996;22:73-9.
 [22] Smith CA, Farrah T, Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 1994;76:959-62.
 [23] Tartaglia LA, Rothe M, Hu YF, Goeddel DV. Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF receptor. Cell 1993;73:213-6.
 [24] Trauth BC, KIas C, Peters AM, Matzku S, Moller P, Falk W, Debatin KM, Krammer PH. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 1989;245:301-5.
[25] Houghton JA. Apoptosis and drug response. CUlT Opin Oncol 1999;11:475-81.
[26] Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer 1999;83:18-29.
[27] Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999;80:827-41.
[28] Ma L, Krishnamachary N, Center MS. Phosphorylation of the multidrug resistance associated protein gene encoded protein P190. Biochemistry 1995;34:3338-43.
[29] Ding Z, Yang X, Chernenko G, Tang SC, Pater A. Human papillomavirus type 16-immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrugresistance phenotype. Int J Cancer 2000;87:818-23.
[30] Tsutsumi K, Belaguli N, Qi S, Michalak TI, Gulliver WP, Pater A, Pater MM. Human papillomavirus 16 DNA immortalizes two types of normal human epithelial cells of the uterine cervix. Am J Pathol 1992;140:255-61.
[31] Yang X, Jin G, Nakao Y, Rahimtula M, Pater MM, Pater A. Malignant transformation of HPV 16-immortalized human endocer- vical cells by cigarette smoke condensate and characterization of multistage carcinogenesis. Int J Cancer 1996;65:338-44.
[32] Yang X, Hao Y, Ding Z, Pater A. BAG-I promotes apoptosis induced by N-(4-hydroxyphenyl)retinamide in human cervical carcinoma cells. Exp Cell Res 2000;256:491-9.
[33] Schmid I, Uittenbogaart CH, Giorgi JV. Sensitive method for measuring apoptosis and cell surface phenotype in human thymo- cytes by flow cytometry. Cytometry 1994;15:12-20.
[34] Vasey PA, Jones NA, Jenkins S, Dive C, Brown R. Cisplatin, camptothecin, and taxol sensitivities of cells with p53-associated multidrug resistance. Mol Pharmacol 1996;50: 1536-40.
[35] Liepins A, Younghusband HB. A possible role for K+ channels in tumor cell injury. Membrane vesicle shedding and nuclear DNA fragmentation. Exp Cell Res 1987;169:385-94.
[36] Yang X, Hao Y, Pater MM, Tang S-C, Pater A. Enhanced expression of anti-apoptotic proteins in human papillomavirus-immortalized and cigarette smoke condensate-transformed human endocervical cells: coITelation with resistance to apoptosis induced by DNA damage. Mol Carcinog 1998;22:95-101.
[37] Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Bioi Chem 1998;273:9357-60.
[38] Cryns V, Yuan J. Proteases to die for. Genes Dev 1998;12:1551-70.

 [39] Ding Z, Yang X, Pater A, Tang SC. Resistance to apoptosis is 430 correlated with the reduced caspase-3 activation and enhanced 431 expression of antiapoptotic proteins in human cervical multidrug- 432 resistant cells. Biochem Biophys Res Cornrnun 2000;270:415-20. 433
 [40] Nunez 0, Benedict MA, Hu Y, Inohara N. Caspases: the proteases of 434 the apoptotic pathway. Oncogene 1998;17:3237-45. 435
 [41] Collins JA, Schandi CA, Young KK, Vesely J, Willingham MC. 436 Major DNA fragmentation is a late event in apoptosis. J Histochem 437 Cytochem 1997;45:923-34. 438
 [42] Weerasinghe P, Hallock S, Liepins A. Bax, bcl-2, and NF-ICB 439 expression in sanguinarine induced bimodal cell death. Exp Mol 440 PathoI2001;71:89-98. 441
 [43] Trump BF, Berezesky IK, Chang SH, Phelps PC. The pathways of 442 cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol 1997;25: 443 82-8. 444
 [44] Majno 0, Joris I. Apoptosis, oncosis and necrosis. An overview of 445 cell death. Am J PathoI1995;146:3-15. 446
 [45] Fernandez-Prada CM, Hoover DL, Tall BD, Venkatesan MM. Human 447 monocyte-derived macrophages infected with virulent Shigella 448 flexneri in vitro undergo a rapid cytolytic event similar to oncosis 449 but not apoptosis. Infect Irnrnun 1997;65:1486-96. 450
 [46] Kuwashima Y. Cytomorphology of murine BI6 melanoma in vivo 451 after treatment with cyclophosphamide: evidence of "oncotic" cell 452 death. Anticancer Res 1996;16:2997-3000. 453
[47] Jonas D, Walev I, Berger T, Liebetrau M, Palmer M, Bhakdi S. Novel 454 path to apoptosis: small transmembrane pores created by staphylo- 455 coccal alpha-toxin in T lymphocytes evoke internucleosomal DNA 456 degradation. Infect Irnrnun 1994;62:1304-12. 457
[48] Matsuoka S, Asano Y, Sano K, Kishimoto H, Yamashita I, Yorifuji H, 458 Utsuyama M, Hirokawa K, Tada T. A novel type of cell death of 459 lymphocytes induced by a monoclonal antibody without participation 460 of complement. J Exp Med 1995;181:2007-15. 461
[49] CUmmings BS, McHowat J, Schnellrnann RO. Phospholipase Azs in 462 cell injury and death. J Pham)acol Exp Ther 2000;294:793-9. 463
[50] Ma F, Zhang C, Prasad KV, Freeman OJ, Schlossman SF. Molecular 464 cloning of ~ a novel cell surface receptor mediating oncotic 465
     cell death. Proc Natl Acad Sci USA 2001;98:9778-83. 466 [51] Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels 467
     determine cell death fate by apoptosis or necrosis. Cancer Res 468
[52] Sapirstein A, Bonventre IV. Specific physiological roles of cytosolic 470 phospholipase Az as defined by gene knockouts. Biochim Biophys 471 Acta 2000;1488:139-48. 472
[53] Zanke BW, Lee C, Arab S, Tannock IF. Death of tumor cells after 473 intracellular acidification is dependent on stress-activated protein 474 kinases (SAPK/JNK) pathway activation and cannot be inhibited by 475 Bcl-2 expression or interleukin 1/3-converting enzyme inhibition. 476 Cancer Res 1998;58:2801-8. 477
[54] Henkels KM, Turchi II. Cisplatin-induced apoptosis proceeds by 478 caspase-3-dependent and -independent pathways in cisplatin-resistant 479 and -sensitive human ovarian cancer cell lines. Cancer Res 480 1999;59:3077-83.
[55] Stefanis L, Park DS, Friedman WJ, Oreene LA. Caspase-dependent 482 and -independent death of camptothecin-treated embryonic cortical 483 neurons. J Neurosci 1999;19:6235-47. 484
[56] Chi S, Kitanaka C, Noguchi K, Mochizuki T, Nagashima Y, Shirouzu 485 M, Fujita H, Yoshida M, Chen W, Asai A, Himeno M, Yokoyama S,
    Kuchino Y. Oncogenic Ras triggers cell suicide through the activation 487 of a caspase-independent cell death program in human cancer cells. 488 Oncogene 1999;18:2281-90. 489
[57] Kitanaka C, Kuchino Y. Caspase-independent programmed cell death 490 with necrotic morphology. Cell Death Differ 1999;6:508-15. 491
[58] Xue L, Fletcher OC, Tolkovsky AM. Autophagy is activated by 492 apoptotic signalling in sympathetic neurons: an alternative mechan- 493 ism of death execution. Mol Cell Neurosci 1999;14:180-98. 494

. Corresponding author. Tel.: +1-709-777-6488; fax: +1-709-777-7010. E-mail address: (A. Pater).
  Abbreviations: PCD, programm~d cell death; COOP, cis-diamminedi- chloroplatinum (II), cisplatin; MDR, multidrug resistance (or resistant); HPV, human papillomavirus; CSC, cigarette smoke condensate; PARP, poly(ADP-ribose) polymerase; BCD, blister cell death/oncosis.

  0006-2952/02/$ - see front matter (j;;) 2002 Published by Elsevier Science 1m PII: SOO06-2952(02)00902-4 Z. Ding et al.IBiochemical Phannacology 7194 (2002)