Abstract:
An extract of  Carica papaya  leaves obtained from a plant of age six months and younger from the date of germination of the seed is used as an inhibitor of HIF to effectively block hypoxia-inducible factor (HIF) function and methods of use thereof. More specifically it relates to the use of the  Carica papaya  plant extract to eliminate unwanted cells by inhibiting HIF in the prevention and treatment of hypoxia-related conditions and diseases such as inflammatory diseases, vascular diseases, cancer and infectious diseases. In a particular embodiment, a solid hypoxic tumor in a patient is treated by administering to the patient a therapeutically acceptable amount of an extract of  Carica papaya  leaves obtained from a plant of age six months and younger from the date of germination of the seed, preferably an alcohol extract such as a methanol extract, whereby to inhibit hypoxia-inducible factors in the tumor.

Description:
[0001]    This application is a divisional application of U.S. Ser. No. 14/208,524 filed Mar. 13, 2014 which claims the benefit of Provisional Patent Application No. 61/784,150, filed Mar. 14, 2013 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to the inhibition of hypoxia-inducible factor activity. 
       BACKGROUND OF THE INVENTION 
       [0003]    The use of plant extracts in Traditional Chinese Medicine (TCM) can be traced back to 6000 years ago (Solecki and Shanidar, 1975). In recent years, there has been a burgeoning use of plant extracts in TCM for modern drug discovery (Graziose et al., 2010). These plant extracts are used for a variety of purposes including health improvement, beauty, and nutritional supplementation, as well as prevention and treatment of diseases, including diabetes and cancer (Graziose et al., 2010). 
         [0004]      Carica papaya  one of the candidate plants. It has been reported to have medicinal properties towards various diseases including dengue fever (Ahmad et al., 2011), wound healing (Mahmood et al., 2005) and asthma (Canini et al., 2007). Extracts from  Carica papaya  were also claimed to have anti-cancer properties (Morimoto et al., US Patent Application No. 20080069907). These investigators, however, did not indicate the specific mechanisms that were involved in the anti-cancer effects, nor did they indicate that the age of the plant could have any effect on the efficacy of the treatment. 
         [0005]    Oxygen homeostasis is a critical element for physiological well-being of the human body. Limited oxygen supply, termed hypoxia, plays a major role in the pathobiology of solid tumors. Hypoxia and oxidative stress pathways are associated with various human disorders, including inflammatory diseases, vascular diseases, cancer and infectious diseases. Cells in hypoxic regions of tumors are more resistant to radiation and chemotherapy (Brown, 2000). They are also more resistant to cell death signaling (Seol et al., 2009). Master regulators of cell survival under hypoxia are the hypoxia-inducible factors (HIFs), HIF-1 and HIF-2. These transcription factors regulate several processes vital for the cells to survive the hypoxic conditions (Semenza, 2011; Miyara et al. 2011). Since these cancer cells have altered metabolic mechanisms for survival under hypoxia, we conjectured that their responses to plant extracts also will be different from normoxic cancer cells. Even though HIF-inhibitory drugs have been approved by the US-FDA for clinical use (Xia et al., 2012), their undesirable side effects are still problematic (Sanchez et al., 2012; Yamaguchi et al., 2012). Due to the limited number of drug candidates in the pipeline and the adverse side effects of the approved ones, identification and development of candidate drug inhibitors that target the HIF-1 pathway are urgently needed. 
       BRIEF SUMMARY OF THE INVENTION 
       [0006]    We have discovered that methanolic extracts of certain  Carica papaya  leaves have a potent inhibitory effect on HIF. The  Carica papaya  leaves effective in this invention are “young” leaves, which in this invention is defined as obtained from a plant of age six months and younger from the date of germination of the seed, i.e., six months and younger from the date the plant seed sprouts. Cytotoxicity analyses showed that such young  Carica papaya  extracts caused high toxicity towards hypoxic cells but not normoxic cells. This specificity is crucial when one targets to eliminate only the hypoxic cells. Based on this specificity, adverse side effects of the extracts and their general toxicity on non-target cells will potentially be eliminated. Therefore, in accordance with this invention, an extract of young  Carica papaya  leaves is used as an inhibitor of HIF to effectively block hypoxia-inducible factor (HIF) function and methods of use thereof. More specifically it relates to the use of such young  Carica papaya  plant extract to eliminate unwanted cells by inhibiting HIF. The compounds and compositions of the present invention are useful in the prevention and treatment of hypoxia-related conditions and diseases such as inflammatory diseases, vascular diseases, cancer and infectious diseases. 
         [0007]    In a particular embodiment, a method is provided for treating a solid hypoxic tumor in a patient, comprising the steps of identifying a patient having a solid hypoxic tumor, and administering to the patient an extract of young  Carica papaya  leaves. The extract can be administered to the patient as a therapeutically acceptable amount of a pharmacon comprising the extract in a pharmaceutically acceptable carrier wherein the patient is not otherwise indicated for treatment with the extract of  Carica papaya  leaves. The pharmacon can be formulated as a liposome, incorporated into a polymer release system, or suspended in a liquid in a dissolved form or as a colloidal form. 
         [0008]    In another embodiment, the extract is administered in combination with a chemotherapeutic agent, such as cisplatin or chetomin. 
         [0009]    In another embodiment of the invention, we provide as a composition of matter an alcohol extraction from young  Carica papaya  leaves. 
         [0010]    In another embodiment of the invention, we provide an article of manufacture comprising a vessel containing an extract of young  Carica papaya  leaves and instructions for use of the extract for the treatment of a solid hypoxic tumor in a method comprising identifying a patient having a solid hypoxic tumor, and administering to the patient an effective amount of an extract of young  Carica papaya  leaves. An effective amount of a composition for treating a cancer will be that amount necessary to inhibit mammalian cancer cell proliferation in situ. Those of ordinary skill in the art are well-schooled in the art of evaluating effective amounts of anti-cancer agents. 
         [0011]    In another embodiment of the invention, we provide an article of manufacture comprising packaging material and contained within the packaging material, an extract of young  Carica papaya  leaves wherein the packaging material comprises a label that indicates that the extract can be used for treating a solid hypoxic tumor 
         [0012]    In a further embodiment of the invention, the young  Carica papaya  leaves are extracted with an alcohol, which can be monohydric, polyhydric, unsaturated aliphatic, or alicyclic 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]    For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which: 
           [0014]      FIG. 1A  shows the effects of methanolic plant extracts on Saos-2 cells in normoxia and hypoxia with dose response curves from an MTT assay and where cisplatin and chetomin were used as positive controls. 
           [0015]      FIG. 1B  shows a compilation of the IC 50 &#39;s of the extracts and experimental control drugs in which data are shown as mean±S.E.M. of triplicates from a representative of three independent experiments, and in which the asterisk (*) represents a significant difference (P&lt;0.05) among the IC 50  in normoxia and hypoxia. 
           [0016]      FIG. 2  shows the effects of plant extract treatment on HIP activities in hypoxic Saos-2 cells where a hypoxia driven firefly luciferase reporter assay was used to measure HIF activity in the cells and positive values indicate increased HIF inhibition while negative values showed HIF activation. 
           [0017]      FIG. 3  shows the effects of  Carica papaya  extract treatment on HIF-driven reporter gene expression in which the HRE-luc Saos2 cells (Malaysian patent filing #: PI 2012003492) were treated with different concentrations of  Carica papaya  extract and incubated in either normoxic or hypoxic conditions, and in which Luciferase signal intensities were measured after 24 hours. 
           [0018]      FIG. 4  shows the percent HIF inhibition in the plant extract or drug-treated hypoxic HRE-luc Saos2 cells where  Carica papaya  and control extract concentrations were 0.1 mg/ml., cisplatin concentration was 0.1 mM. The percent HIF inhibition was calculated as a ratio of the difference between untreated and treated sample to the untreated sample, and a positive value indicates HIF inhibition, while a negative value denotes HIF activation. 
           [0019]      FIG. 5  shows the percent of HIF inhibition for leave crude extracts obtained from plants of age either six months or younger or older than six months, where negative inhibition value indicates activation of HIF, the symbol “*’ denotes statistically significant difference (P&lt;0.05) compared to the untreated control, B=Batch number, and R=Replicate. 
           [0020]      FIG. 6  shows the percentage of HIF inhibition with brew samples using prior art extraction procedures described in Morimoto et al. US patent Application No. 20080069907 on leaves obtained from plants older than 10 months from the date of germination of the seed, where negative inhibition value indicates activation of HIF, B=Batch number, and R=Replicate. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    The discovery of the unique properties of the extract of young  Carica papaya  leaves arose from an investigation of the effects of the extracts of various plants on HIF activity, and the effect of the age of the  Carica papaya  leaves on HIF inhibitory activities. Descriptions of those experiments will be given followed by specific investigations of extracts from  Carica papaya  leaves. 
       The Effects of the Extracts of Various Plants on HIP Activity 
     Cell Line and Culture Conditions 
       [0022]    A human osteosarcoma cell line, Saos-2 stably expressing four copies of the erythropoietin hypoxia response elements (HRE-luc Saos2; Shafee et al., Malaysian Patent Application #: PI 2012003492), was cultured in DMEM medium (PAA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; PAA, USA) and 1% (v/v) antibiotic-antimycotic (PAA, USA) in a humidified incubator supplied with ambient oxygen and 5% CO 2  at 37° C. An hypoxic gaseous environment was created by incubating cells in a ProOx in vitro chamber (BioSpherix), controlled by ProOx model 110 (BioSpherix). The cells were supplied 0.5% O 2 , 94.5% N 2  and 5% CO 2  at 37° C. 
         [0000]    ////
 
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       Plant Materials, Cell Culture Treatment and Viability Assay 
       [0023]    Nine types of plants with known medicinal properties were tested in this study, and are described in Table 1: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Plants used in the study and their medicinal uses. 
               
             
          
           
               
                 Plant name 
                 Medicinal use and references 
               
               
                   
               
               
                 Melastomataceae 
                 Anti diarrhoeal (Sunilson et al., 2009), antimicrobial (Thatoi et 
               
               
                 
                   Melastoma 
                 
                 al., 2008), cytotoxicity, antiviral (Lohezic Le Devehat et al., 
               
               
                 
                   malabathricum 
                 
                 2002), anticoagulant (Manicam et al., 2010), antipyretic, 
               
               
                 L. 
                 anti-inflammatory, antinociceptive (Zakaria et al., 2006), 
               
               
                   
                 antioxidant, free radical scavenging activity and anticancer 
               
               
                   
                 (Susanti et al., 2007). 
               
               
                 Acanthaceae 
                 Antidiabetic, diuretic, antilytic, laxative (Sunarto, 1977), 
               
               
                 
                   Strobilanthes 
                 
                 anticancer (Yaacob et al., 2010), anti-AIDS, antihypertensive 
               
               
                 
                   crispa 
                 
                 (Liza et al, 2010), antioxidant (Abu et al., 2007), 
               
               
                 T. Anderson 
                 antihyperglycemic and antilipidemic (Fadzelly et al., 2006) 
               
               
                 Cactaceae 
                 Antitumour, anti-rheumatic, antiulcer, anti-inflammatory 
               
               
                 
                   Pereskia 
                 
                 (Goh, 2000), relief of headache, gastric pain, hemorrhoids, 
               
               
                 
                   grandifolia 
                 
                 atopic dermatitis, body revitalization (Goh, 2002; Rahmat, 
               
               
                 Haw. 
                 2004; Tan et al., 2005), reduce swelling (Sahu et al., 1974; 
               
               
                   
                 Anon, 1969) and antioxidant (Sim et al., 2010). 
               
               
                 Compositae 
                 Eruptive fever, rash, kidney disease, migraines, constipation, 
               
               
                 
                   Gynura 
                 
                 hypertension, diabetes mellitus, cancer (Perry, 1980), 
               
               
                 
                   procumbens 
                 
                 anti-herpes simplex virus, antidiabetic, urinary infection, 
               
               
                 (Lour.) Merr. 
                 antiallergic, reduce cholesterol and high blood pressure 
               
               
                   
                 (Jiratchariyakul et al., 2000), antihyperglycaemic (Akowuah et 
               
               
                   
                 al., 2001; Akowuah et al., 2002; Hassan et al., 2010), 
               
               
                   
                 anti-inflammatory (Iskander et al., 2002; Jiratchariyakul et al., 
               
               
                   
                 2000), anti-hyperlipidaemic (Zhang and Tan, 2000), 
               
               
                   
                 hypertension (Lam et al., 1998; Kim et al., 2006), wound 
               
               
                   
                 healing (Zahra et al., 2011) and antiulcer (Mahmood et al., 
               
               
                   
                 2010), antioxidant and antitumour (Maw et al., 2011) 
               
               
                 Umbelliferae 
                 Cancer (Yu et al., 2007; Huang et al., 2008), fever 
               
               
                 
                   Hydrocotyle 
                 
                 (Manandhar, 1993; Pfoze et al., 2012), edema, detoxication, 
               
               
                 
                   sibthorpioides 
                 
                 throat pain, diuretic, psoriasis (Li, 1986), rheumatalgia, 
               
               
                 Lam. 
                 dysentery, whooping cough, jaundice (Srivastava et al., 2012), 
               
               
                   
                 viral infection (Li, 2000; Wang 2000; Yu et al., 2007). 
               
               
                 Caricaceae 
                 Anti-tumor (Otsuki et al., 2010), anti-Dengue fever (Ahmad 
               
               
                 
                   Carica 
                 
                 et al., 2011), antisickling (Imaga et al., 2009), vasodilatatory, 
               
               
                 
                   papaya 
                 
                 antioxidant, reduction of cardiovascular risks (Runnie et al., 
               
               
                 L. 
                 2004), wound healing (Mahmood et al., 2005), asthma 
               
               
                   
                 relieving, vermifuge, treatment of gastric problems, fever, 
               
               
                   
                 amoebic dysentery (Canini et al., 2007). 
               
               
                 Labiatae 
                 Antioxidant (Hsu et al., 2010), anti-inflammatory (Hsu et al., 
               
               
                 
                   Orthosiphon 
                 
                 2010); Nadia et al., 2012), kidney stones (Hsu et al., 2010), 
               
               
                 
                   aristatus 
                 
                 dysuria (Premgamone et al., 2008), hypertension (Matsubara 
               
               
                 (Blume) Miq. 
                 et al., 1999; Ohashi et al., 2000). 
               
               
                 Cactaceae 
                 Anticancer (Malek et al., 2009; Tan et al., 2005), antitumour, 
               
               
                 
                   Pereskia 
                 
                 anti-rheumatic, antiulcer, anti-inflammatory, antidiabetic and 
               
               
                 
                   bleo 
                 
                 hypertension (Goh, 2000), antiproliferative and mutagenic (Er 
               
               
                 DC. 
                 at al., 2007), relief of headache, gastric pain, hemorrhoids, 
               
               
                   
                 atopic dermatitis, body revitalization (Goh, 2002; Rahmat, 
               
               
                   
                 2004; Tan et al., 2005). 
               
               
                 Acanthaceae 
                 Skin rashes, insect and snake-bite, analgesic, anti- 
               
               
                 
                   Clinacanthus 
                 
                 inflammatory (Satayavivadetal., 1996; Wanikiat et al., 2008), 
               
               
                 
                   nutans 
                 
                 anti-HSV and anti-VZS (Jayavasu et al, 1992; Thawaranantha 
               
               
                 (Burm. f.) Lindau 
                 et al., 1992), bladder activity (Low et al., 2011). 
               
               
                   
               
             
          
         
       
     
         [0024]    The voucher specimens were deposited in the Herbarium of Institute of Biosciences, Universiti Putra Malaysia. Leaves of these plants were subjected to methanolic extraction as described in Hsu et al., 2010. The leaves were washed with distilled water and left to air-dry at room temperature or in an oven at 40° C. until a constant weight was obtained. Dried leaves were then blended into a powder form, soaked and extracted in methanol at a 1:3 ratio (w/v) for three days at room temperature. Extracts were filtered by Whatman No. 1 filter paper with the aid of a vacuum pump. The residues were re-extracted twice using the same methanolic method. Filtrates were then concentrated by rotary evaporator at a maximum of 40° C. Concentrated extracts were collected in glass vials with push-in caps and dried in an oven at 40° C. until a constant weight was obtained. All of the crude extracts were stored −80° C. Prior to use, the extracts were prepared at the required concentrations by dissolving them in serum-free DMEM media with a final DMSO concentration of less than 0.5% (v/v), centrifuged at 1000×g and filtered through 0.22 μM filters. Overnight cultures of cells, initially seeded at 1.5×10 4  cells per well in a 96-well plate, were treated with selected concentrations of plant extracts and incubated in normoxic or hypoxic conditions. After 24 h of incubation, an MTT assay (Jamal et al., 2012) was employed to determine cell viability following treatment of cultured cells with selected concentrations of the crude plant extract. These concentrations were determined empirically and selected to cover a range of effects, ranging from no effect to significant cytotoxicity. Initially, cells were seeded at 1.5×10 4  cells per well in a 96-well plate for 24 h. The cells were treated with selected concentrations of the extract and incubated in normoxic or hypoxic conditions. After 24 h of treatment, spent culture medium was replaced with fresh serum-free DMEM containing 0.5 mg/ml MTT. After a further 4 h of incubation in 5% CO 2  at 37° C., formazan precipitates that formed were dissolved with 100 μl of DMSO and the reaction was read in a microplate reader (Model 550, BioRad, Hercules, Calif.) at 570 nm absorbance and 630 nm as the reference wavelength. 
       HIF Reporter Assay 
       [0025]    Cells were co-transfected with a hypoxia-driven firefly luciferase reporter plasmid construct containing four copies of the erythropoietin (EPO) hypoxia response elements (HRE) and a pRL-CMV expressing  Renilla  luciferase as described previously (Kaluz et al., 2008, Shafee et al., 2009, Shafe et al. Malaysian Patent Application No. PI 2012003492). Transfected cells were treated with appropriate concentrations of plant extracts and incubated in either normoxic or hypoxic conditions. After 24 h, firefly and  Renilla  luciferase signal intensities were measured (Kaluz et al., 2008, Shafee et al, 2009). Percent HIF inhibition was calculated as a ratio of the difference between untreated and plant extract-treated sample to the untreated sample. A positive value indicates HIF inhibition, while a negative value denotes HIF activation. 
       Statistical Analysis 
       [0026]    The Student t-test was used to analyze the experimental data in this study. Results were expressed as mean±standard error of the mean (SEM). A ρ value of &lt;0.05 was considered significant. 
         [0000]    ////
 
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       Results and Discussion 
       [0027]    To investigate whether hypoxia affects cellular responses to plant extract treatment, cells were treated with selected concentrations of each extract, and their viability was determined. As positive controls for inhibition of HIF activity, we included cisplatin (Duyndam et al., 2007), and Chetomin (Tan et al., 2005). Different patterns of cytotoxicity were observed when the cells were treated in normoxic versus hypoxic conditions. As expected (Song et al., 2006), the IC 50  value for cisplatin in hypoxia is significantly higher than in normoxia ( FIG. 1A ). For the first time, we show that the IC 50  of chetomin also increased in hypoxia. 
         [0028]      Melastoma malabathricum, Strobilanthes crispus and Pereskia grandifolia , showed no drastic differences in cytotoxicity when lower concentrations (&lt;50 μg/ml) of extracts were used ( FIG. 1A ). However, at concentrations higher than 150 μg/ml, cytotoxicity to  Melastoma malabathricum  became more evident in the hypoxic compared to the normoxic cultures. Cytotoxic and antiproliferative activities of methanolic extracts of  Melastoma malabathricum  have been previously reported (Devehat et al., 2002). Their studies, which were done under normoxia, showed that the IC 50  values of the extract ranges from 19 to &gt;400 μg/ml depending on the cell line tested. Results in the present study showed that hypoxic cancer cells are more susceptible to  Melastoma malabathricum  cytotoxicity when the concentrations used are above 150 μg/ml. Therefore, it is likely that the IC 50  of the extracts will be lower in hypoxic cancer cells. For  Strobilanthes crispus  the difference in cytotoxicity began to be seen as early as 100 μg/ml. Beginning at this concentration, cells in the hypoxic environment showed a higher rate of cell death compared to the cells in the normoxic environment. The opposite situation was observed in the cells treated with  Pereskia grandifolia . Cytotoxicity of the cells was observed to be higher in the normoxic condition instead, even though cytotoxicity was seen earlier, at 25 μg/ml. 
         [0029]      Gynura procumbens, Hydrocotyle sibthorpioides  and  Carica papaya  extracts showed different cytotoxicities in normoxia and hypoxia at all concentrations used.  Gynura procumbent  and  Hydocotyle sibthorpioides  were found to induce cell proliferation at concentrations lower than 150 μg/ml. No induction was seen for  Carica papaya  extract. Interestingly, in hypoxia, all these extracts induced higher cell proliferation than in normoxia. To the best of our knowledge, this is the first report to show induction of cell proliferation in cancer cells by these three plant extracts.  Gynura procumbens  has been shown to contribute towards the wound healing process (Zahra et al., 2011). Our findings of increased cell proliferation, albeit in cancer cells, may help contribute towards further understanding of mechanisms involve in the process of wound healing. Similar to our findings in the normoxic condition, the inefficiency of  Hydrocotyle sibthorpioides  extracts in cell killing was also reported previously (Huang et al., 2008). They showed that the IC 50  of  Hydrocotyle sibthorpioides  ethanolic extracts in cancer cells was &gt;2000 μg/mL. For  Carica papaya , we observed a minimal stimulatory effect in hypoxia at concentrations lower than 250 μg/ml. However, above this concentration, its cytotoxicity increased tremendously. In normoxia, on the other hand, the extract showed a gradual increase in cytotoxicity with increasing concentrations of extracts. This result is supported by Otsuki et al. (2010) which reported anti-proliferative responses of various tumor cell lines in normoxic conditions towards  Carica papaya  extracts. 
         [0030]      Orthosiphon aristatus, Pereskia bleo  and  Clinacanthus nutans  extracts showed minimal cytotoxicity in both normoxic and hypoxic conditions.  Orthosiphon aristatus  extract, which was previously shown to have antioxidant and anti-inflammatory effects (Hsu et al., 2010), showed a gradual reduction of cell viability with increasing amounts of extract used in the normoxic condition. But in the hypoxic condition, no statistically significant difference in cytotoxicity was observed until 500 μg/ml were used, when a sharp drop in cell viability was observed in both normoxic and hypoxic conditions, indicating a general cytotoxity. For  Pereskia bleo  and  Clinacanthus nutans  extracts, no significant cytotoxicity was seen until at the highest concentrations tested under normoxic conditions. This finding is in agreement with a study by Er et al. (2007) which also failed to observe any notable anti-proliferative effect of  Pereskia bleo  methanolic extract in 4T1 and NIH/3T3 cell lines. In contrast, Malek et al. (2009) reported cytotoxicity effects of  Pereskia bleo  methanolic extract in several cancer cell lines. Besides the different types of cancer cells used, another possible explanation for these inconsistencies is the gaseous conditions used in their studies. In our study, we found that hypoxic environment led to growth stimulatory response by  Pereskia bleo  but not for  Clinacanthus nutans . This result strongly suggests that microenvironmental conditions contribute towards cellular responses to plant extract treatments. 
         [0000]    Specific Investigation of Extracts from  Carica papaya  Leaves. 
         [0031]    The following describes the specific investigation of whether HIF is affected by  Carica papaya  plant extract treatment, HRE-luc Saos2 cells were treated with selected concentrations of the extract, and their HIF-responsive luciferase signal was determined. The untreated samples showed the expected response pattern, where the signal was only increased in hypoxia but not normoxia ( FIG. 3 ). The high hypoxic signal was dramatically reduced in cells treated with various concentrations of  Carica papaya  extracts. At the lowest concentration tested (0.1 mg/ml) the signal was reduced by almost 40%. As the concentration of the extract increased, the signal also decreased. However, due to the possibility of general cytotoxicity of the extracts on the cells, the reduced signals need to be further studied. Prior to performing further investigation, we compared the inhibitory properties of the  Carica papaya  against cisplatin, a known inhibitor of HIF (Duyndam et al., 2007). 
         [0032]    At 0.1 mg/ml concentration,  Carica papaya  extract reproducibly inhibits HIP signals by approximately 40% ( FIG. 4 ). This inhibition was comparable to cisplatin, where the inhibition was around 60%. To confirm that the signal inhibition was not due to a general effect of extract treatment on the HRE-luc Saos2 cells, another plant extract was used as a control. Treatment of this extract at a similar concentration to  Carica papaya  extract, did not result in any HIF inhibitory signal. Instead, a slight increase was observed. This increase was expected since HIF is also a stress-response signal (Semenza, 2011; Miyata et al., 2011). Therefore, cells which undergo stress, in this case perhaps by the presence of the extract, will display a slight increase in HIF activity. These data clearly demonstrate the specificity of  Carica papaya  extract on HIF inhibition. 
         [0033]    To verify that the reduced HIF signals were not due to cytotoxicity effects of the  Carica papaya  extract on the cells, we performed an MTT assay on the treated cells. At 0.1 mg/ml concentration, where HIF signal was suppressed by as much as 40%, the cells appeared to be viable ( FIG. 1A ). In fact, the cell number was slightly increased in the hypoxic condition, suggesting a minimal effect of the  Carica papaya  extract on the general properties of the cells. At higher concentrations, the extract began to affect cell viability, particularly in the hypoxic condition. This interesting observation suggests that hypoxic cells are more susceptible to  Carica papaya -induced cytotoxicity compared to normoxic cells. Therefore,  Carica papaya  is an ideal candidate to eliminate unwanted hypoxic cells. To observe the pattern of HRE-luc Saos2 cell killing by HIF-inhibitors in normoxic and hypoxic conditions, cisplatin was used. Additionally, another specific inhibitor of HIF, chetomin (Tan et al., 2005), was also used. This was a proof-of-concept study to confirm the results observed in the  Carica papaya  extract-treated samples. Referring to the respective plots for  Carica papaya , cisplatin, and chetomin in  FIG. 1A , a different pattern of cytotoxicity was observed when the cells were treated with these drugs in normoxic versus hypoxic conditions. As expected, the IC 50  value for cisplatin in hypoxia is significantly higher than in normoxia. Hypoxia-induced resistance to cisplatin treatment was previously reported (Song et al., 2006). For the first time, we have shown in this study that the IC 50  of chetomin also increased in hypoxia. 
       Overall 
       [0034]    Our data demonstrate that methanolic  Carica papaya  plant extracts showed different IC 50  values in hypoxic versus normoxic conditions ( FIG. 4 ). This is not entirely unexpected, since cellular responses and adaptations to the hypoxic environment are complex and play important roles in normal cellular physiology. Hypoxic tumor cells are known to be more resistant to certain drugs (Reviewed in Brown, 2000). We found that the IC 50  of  Carica papaya  extract in hypoxia was reduced by almost 3-fold when compared to normoxia. This finding demonstrates  Carica papaya  as a candidate for the elimination of unwanted hypoxic cells. To investigate the effects of a combination treatment, using  Carica papaya  with either cisplatin or chetomin, we performed experiments using IC 50  concentrations of each material. Results obtained showed that the combination treatment led to an increase in cytotoxicity compared to individual treatments. In this combination treatment, the killing effect improved by 4-fold in both conditions tested. This observation suggests that  Carica papaya  is a good candidate, as individual or combination treatment for drug resistant cells, particularly with respect to targeting hypoxic cells. 
         [0035]    Since HIF is known to be a master regulator of cellular responses to low oxygen conditions (Semenza, 2011), it is likely that the variation in cellular responses to plant extracts in hypoxic versus normoxic conditions was due to differences in their HIP activities. To investigate this possibility, a hypoxia-driven reporter assay (Kaluz et al., 2008) was performed in samples treated with the lowest concentrations of each plant extract. Signal intensity of this assay is directly proportional to HIF activity. These lowest concentrations were chosen since they provide the least effects on the general properties of the cells. Treatment of cells with plant extracts did not show any significant variation in the normoxic basal level of HIF activities (data not shown). Varying responses, however, were observed in the hypoxic samples ( FIG. 2 ). Treatment with all of the plant extracts, except for  Carica papaya , resulted in further activation of HIP activity, as evidenced from the negative values of the HIF inhibition. The level of activation however, varied among the different plant extracts. These variations did not show any obvious correlation to their viability at the low concentrations tested ( FIG. 1A ). Interestingly, the only candidate HIP Inhibitor found in this study, which was the  Carica papaya  extract, led to an almost 40% inhibition of HIP activity. This inhibition was almost comparable to cisplatin treatment, a known HIF inhibitor (Duyndam et al., 2007). This novel finding indicates the potential of  Carica papaya  extract as an agent to kill hypoxic cancer cells through inhibition of HIF activity. 
         [0036]    Data presented in the foregoing study show specific cytotoxic effects of  Carica papaya  extract under hypoxic conditions as evidenced from a 3-fold reduction in IC 50  of hypoxic versus normoxic cells. This reduced IC 50  was achieved through specific inhibition of HIF activities. 
         [0037]    Having established the effectiveness of  Carica papaya  extract to inhibit HIP activity, experiments were performed to determine the effect the age of the  Carica papaya  leaves on HIF inhibition. Leaves of the  C. papaya  were subjected to the methanolic extraction method to obtain crude leave extracts. Several batches of leave samples were originally obtained from different ages of plants from a farm that cultivates specific  C. papaya  varieties. The extracts were used in a HIF inhibition assay using the cell-based HIF assay system described above. 
         [0038]    HHIF inhibitory activities were found to be dependent on the age of the plant from where the leaves were originally harvested. Referring to  FIG. 5 , statistically significant, replicable inhibition was seen in crude extract of leaves from plants ages 6 months and younger. An opposite effect, statistically significant activation of HIF, was noted when samples were obtained from plants that were older than 6 months of age. 
         [0039]    To compare the invention to the disclosure in prior art Morimoto et al. US patent Application No. 20080069907, HIF assays were performed using extraction procedures detailed in their patent application with plants that were older than 10 month. Referring to  FIG. 6 , results showed that a replicable inhibition of HIF was not seen. The HIF activity was instead increased, as evident from the negative values of HIF inhibition (the y-axis). 
         [0040]    The alcohol used to extract the  Carica papaya  leaves was methanol. In general, one can use any of the monohydric, polyhydric, unsaturated aliphatic, or alicyclic alcohols, exemplified by methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, pentan-1-ol, 3-methylbutan-1-ol, 2-methylbutan-1-ol, 2,2-dimethylpropan-2-ol, pentan-3-ol, pentan-2-ol, 3-methylbutan-2-ol, 2-methylbutan-2-ol, ethane-1,2-diol, propane-1,2-diol, propane-1,2,3-triol, butane-1,2,3,4-tetraol, or pentane-1,2,3,4,5-pentol. The alcohol is preferably an acyclic alcohol having from 1 to five carbon atoms, most preferably methanol. 
         [0041]    As described above, performed experiments using combinations of  Carica papaya  and cisplatin or chetomin led to an increase in cytotoxicity by 4-fold. Other chemotherapeutic agents can be used. More particularly, one can use in combination with the  Carica papaya  extract a chemotherapeutic agent selected from the group consisting of cisplatin, chetomin, methotrexate, trimetrexate, adriamycin, taxotere, 5-fluorouracil, vincristine, vinblastine, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol, tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, 0-epi-1,25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate, brequinar sodium, bropirimine, budotitane, busulfan, buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone, camptothecin derivatives, canarypox IL-2, caracemide, carbetimer, carboplatin, carboxamide-amino-triazole, carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derived inhibitor, carubicin hydrochloride, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine, cytarabine ocfosfate, cytotytic factor, cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox, diethylnorspermnine, dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docosanol, dolasetron, doxifluridine, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, dronabinol, duazomycin, duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolomab, eflomithine, eflornithine hydrochloride, elemene, elsamitrucin, emitefur, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, epristeride, erbulozole, erythrocyte gene therapy vector system, esorubicin hydrochloride, estramustine, estramustine analog, estramustine phosphate sodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorocitabine, forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine hydrochloride, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor-1 receptor inhibitor, interferon agonists, interferon alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA, interferon gamma-IB, interferons, interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan, irinotecan hydrochloride, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanrmeotide, lanreotide acetate, leinamycin, lenograstim, lentinan sulfate, leptolstatin, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole, liarozole, liarozole hydrochloride, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate sodium, metoclopramide, metoprine, meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, mycophenolic acid, myriaporone, n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, nocodazol, nogalamycin, n-substituted benzamides, 06-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole, peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin, piritrexim, piroxantrone hydrochloride, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, puromycin hydrochloride, purpurins, pyrazofurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes, RII retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDII mimetics, semustine, senescence derived inhibitor I, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, simtrazene, single chain antigen binding protein, sizofuran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoictin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tropisetron, tubulozole hydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin stimalamer, and zorubicin hydrochloride, as well as salts, homologs, analogs, derivatives, enantiomers and/or functionally equivalent compositions thereof. 
         [0042]    Although the present invention has been described in connection with the preferred embodiments, it is to be understood that modifications and variations may be utilized without departing from the principles and scope of the invention, as those skilled in the art will readily understand. 
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